Run-in behavior of amorphous carbon films significantly affected their tribological properties. Generally, for amorphous carbon films, a low friction coefficient was obtained after a run-in period during which the friction coefficient was high and gradually decreased to a low value under vacuum. However, in this paper, the friction coefficient was initially low and gradually increased to a high value during run-in period under vacuum. According to experimental results, first principles calculations were selected to probe the possible mechanism of the anomalous run-in behavior. The results showed that the weak and strong atomic interactions should be attributed to low and high friction.