The MARC Penn Line is the primary and busiest of the three MARC Rail Lines. The line operates on the southernmost 76 miles of the electrified Northeast Corridor from Perryville, Maryland, through Baltimore to Washington-Union Station.

The portion of the line between Washington and Baltimore is the only MARC Line that operates throughout the day on weekdays with hourly or better service. This portion of the line is also the only MARC Line with weekend service with 9 round-trips between Washington and Baltimore on Saturdays and 6 on Sundays. There are 3 trains on Saturdays and 2 on Sundays that begin at Martin State Airport in the mornings and return in the evenings for access to the train yard at this station. These weekend trains all skip Seabrook that is only open on weekdays.

Service to and from Perryville is more limited and is provided during weekdays. This service that debuted originally in 1991 was originally called the“Susquehanna Flyer.” Service consists of only seven terminating and six originating MARC Trains. The first two originating trains of the day deadhead from the closest train yard at Martin State Airport to the station before the next four round-trips layover at the Perryville Station with layovers of twenty to thirty minutes. The final three evening northbound trips arrive at the station and then deadhead back to the yard at Martin State Airport.

Martin State Airport receives slightly more weekday service (plus the already mentioned limited weekend service) with two more trains originating at the station during weekdays, and four more trains at the end of both rush hours terminating at the station.

Train consists are normally push-pull diesel trains operating under wire. These trains operate with a mixture of MARC II and IV single-level and bilevel trains. A few Electric HHP-8s are still in service, primarily used for MARC express trains that are the fastest Commuter Rail operations in the United States, reaching speeds of 125mph. These units were supposed to be replaced by Siemens Charger SC-44 locomotives in 2018 but were spared from scrapping at the last minute when MARC decided it wasn't a sensible idea to exclusively convert the former electric operations on the Penn Line (MARC previously owned four AEM-7s) to diesel trains.

Commuter-oriented rail service has operated continuously on the Northeast Corridor between Washington and Baltimore since the lines were completed in the 19th Century. This service was originally operated by the Pennsylvania Railroad, followed by its absorption by Penn Central in 1968 and then Conrail starting in 1976. After electrification was completed in the 1930s, trains were originally normal Penn Railroad MP54 EMU units (also used in Philadelphia, New Jersey, and via third rail on the Long Island Rail Road). Service was transitioned to Arrow cars, leased from the NJDOT in the late 1970s to replace the MP54s before being converted to electric push-pull trains (using AEM7 locomotives) in the late 1980s.

In the 1980s the line's name changed twice as well. It was branded as Conrail until 1983 when the Northeast Rail Service Act of 1981 allowed Conrail to stop operating unprofitable Commuter Rail operations. On January 1, 1983 (the same day NJ Transit Rail Operations and Metro-North were born) MDOT began contracting with Amtrak to run the service originally as AMDOT (Amtrak Maryland Department of Transportation), this was later renamed MARC (short for MAryland Rail Commuter, or Maryland Area Rail Commuter) in 1984. This new name also applied to the then B & O-operated but now subsided Camden and Brunswick Commuter Rail lines.

To this day the MARC Penn Line is operated by Amtrak under contract. This relationship means that Amtrak Ticket agents can sell MARC tickets, all MARC trains appear on Amtrak departure boards (properly, not weirdly like SEPTA at Wilmington, DE) and LED Station information signs. Until 2018 MARC even sold their tickets on Amtrak ticket stock, using the now previous generation (as of 2025) Amtrak QuikTrak Machines with MARC tickets sold as paper value but not eTickets. In 2018 MARC introduced their own TVMs and tickets can now be purchased digitally through the CharmPass App.

MARC weekly and monthly tickets can also be used for free travel on Montgomery County RIDE ON and Frederick County TransIT, MTA Local Bus, Light Rail, Baltimore Metro Subway and Neighborhood Shuttles; WMATA Metrobus (base fare only) in Maryland, Washington, DC and Virginia.