Carbon films with two different kinds of sp2 nanocrystallited structure were investigated to study the stick-slip friction with the in-situ and ex-situ tests. In-situ transmission electron microscope (TEM) observation and nanofriction tests revealed that the origins of stick and slip varied with shear stress and film deformation. At the stick stage, shear stress gradually increased with the contact strengthened until reached the shear strength to break the interfacial adhesion; at the slip stage, the shear stress decreased and accompanied with film deformation. During the sliding process, adhesive deformation resulted in the large stick-slip step while ploughing deformation led to a smoother step. Ex-situ nanofriction tests on a series of sp2 nanocrystallited carbon films with different irradiation energies showed the expected sliding behavior with the in-situ results. This study first clarified the mechanism of stick-slip friction with the in-situ TEM observation, which plays the important role for the micro and nano application of sp2 nanocrystallited carbon films.