Astrocytes play a major role in the reactive processes in response to neuronal injuries in the brain. Excessive gliosis is detrimental and can contribute to neuronal damage. CD81 (TAPA), a member of the tetraspanin family of proteins, is upregulated by astrocytes after traumatic injury to the rat central nervous system (CNS). To further understand the role of CD81 in the inhibition of astrocytes, we analyzed the effects of a CD81 antibody, on cultured rat astrocytes. The results indicated that the effect worked in a dose-dependent manner with certain dosage range. It, however, reached a dosage equilibrium at a high dosage. Furthermore, anti-CD81 antibody remarkably inhibited the proliferation of astrocytes after incubation with astrocytes for different periods of time and the effect presented a time-dependent fashion. However, anti-CD81 antibody substantially inhibited the proliferation of astrocytes at low density and middle density but slightly inhibited the proliferation of astrocytes at high density, suggesting that the effect was positively correlated with the proliferative ability of astrocytes. Finally, the cell cycle of astrocytes exposured to anti-CD81 antibody was arrested in S phase at the initial stage and at G(0)/G(1) phase over time. These findings indicated that CD81 exert significant inhibitory effect, dose-dependently and time-dependently, on the proliferation of astrocytes and the effect is positively correlated with the proliferative capability of astrocytes.