CD56+ T cells, the crucial component of the host innate immune system, play an important role in defense against viral infections. We investigated the noncytolytic anti-HIV-1 activity of primary CD56+ T cells. SNs collected from CD56+ T cell cultures inhibited HIV-1 infection and replication. This CD56+ T SN-mediated anti-HIV-1 activity was broad-spectrum, as CD56+ T SNs could inhibit infections by laboratory-adapted and clinical strains of HIV-1. The antibody to IFN-γ could partially block the CD56+ T SN-mediated anti-HIV effect. Investigation of mechanism(s) of the CD56+ T cell action on HIV-1 showed that although CD56+ T SN had little effect on HIV-1 entry coreceptor CCR5 expression, CD56+ T SN induced the expression of CC-chemokines, the ligands for CCR5. The antibodies to CC-chemokines also significantly blocked CD56+ T SN-mediated anti-HIV activity. Furthermore, CD56+ T SN up-regulated the expression of STAT-1/-2 and enhanced the expression of IRF1, -3, -7, and -9, resulting in the induction of endogenous IFN-α/β expression in macrophages. Moreover, CD56+ T SN up-regulated intracellular expression of APOBEC3G/3F, the recently identified HIV-1 restriction factors. These findings provide compelling evidence that CD56+ T cells may have a critical role in innate immunity against HIV-1 infection.