He is best known for his role in the 1903 silent short The Great Train Robbery, which the American Film Institute and many film historians and critics recognize as the production that first established both the Western and action genres, setting a new "narrative standard" in the motion picture industry.

In that film's memorable ending, Barnes points his pistol at the camera and slowly fires all six shots at the viewer.

He played Ham Peggotty in David Copperfield, the earliest known film adaption of the 1850 novel by Charles Dickens.

[4] He also played supporting roles in Nicholas Nickleby (1912), Aurora Floyd (1912), and A Dog of Flanders (1914).

Barnes made his final onscreen appearance for the Edison Studio in Cy Whittaker's Ward, in 1917.