MoS2 film deposited was deposited by closed-field unbalanced-magnetron sputtering (CFUBMS), and its structural and tribological properties were modified by the heat treatment at temperature range of 100–500 °C in vacuum environment. Results revealed that as-deposited MoS2 film was composed of MoS2 plus a little MoO3 with a dense amorphous-like structure. After the vacuum heat treatment, MoS2 film was still very compact, but its microstructure was changed from amorphous-like to nano-crystalline states with the increase of heat treatment temperature. As a result, a MoS2 film with a microstructure of MoS2 nanocrystals embedded into its own amorphous matrix was obtained at middle temperature of 200 °C. Furthermore, MoO3 phase in the film was reduced to MoO2 after the vacuum heat treatment at high temperatures of 400 and 500 °C. Friction tests revealed that the wear resistance of MoS2 film in vacuum environment was significantly improved by the vacuum heat treatment at suitable temperature. The wear life of vacuum-heat-treated MoS2 film at 200 °C for 3 h was ~6.2 × 105 r, about four times longer than that of as-deposited MoS2 film.