The human cytidine deaminase APOBEC3G (A3G) is a part of a cellular defense system against human immunodeficiency virus type 1 (HIV-1) and other retroviruses. Antiretroviral activity of A3G can be severely blunted in the presence of the HIV-1 protein Vif. However, in some cells expressing the enzymatically active low-molecular-mass form of A3G, HIV-1 replication is restricted at preintegration steps, before accumulation of Vif. Here, we show that A3G can be secreted by cells in exosomes that confer resistance to both vif-defective and wild-type HIV-1 in exosome recipient cells. Our results also suggest that A3G is the major exosomal component responsible for the anti-HIV-1 activity of exosomes. However, enzymatic activity of encapsidated A3G does not correlate with the observed limited cytidine deamination in HIV-1 DNA, suggesting that A3G-laden exosomes restrict HIV-1 through a nonenzymatic mechanism. Real-time PCR quantitation demonstrated that A3G exosomes reduce accumulation of HIV-1 reverse transcription products and steady-state levels of HIV-1 Gag and Vif proteins. Our findings suggest that A3G exosomes could be developed into a novel class of anti-HIV-1 therapeutics.