Cell-secreted miRNAs are highly stable and can serve as biomarkers for various diseases and signaling molecules in intercellular communication. The mechanism underlying the stability of circulating miRNAs, however, remains incompletely understood. Here we show that Argonaute 2 (Ago2) complexes and microvesicles (MVs) provide specific and non-specific protection for miRNA in cell-secreted MVs, respectively. First, the resistance of MV-encapsulated miRNAs to RNaseA was both depended on intact vesicular structure of MVs and protease-sensitive. Second, an immunoprecipitation assay using a probe complementary to human miR-16, a miRNA primarily located in the MVs and showed a strong, protease-sensitive resistance to RNaseA, identified Ago2 as a major miR-16-associated protein. Compared with protein-free miR-16, Ago2-associated miR-16 was resistant to RNaseA in a dose- and time-dependent fashion. Third, when the miR-16/Ago2 complex was disrupted by trypaflavine, the resistance of miR-16 to RNaseA was decreased. In contrast, when the association of miR-16 with the Ago2 complexes was increased during cell apoptosis, although the total amount of miR-16 and Ago2 remained unchanged, the resistance of miR-16 to RNaseA in the MVs was enhanced. A similar correlation between the increase of miR-223/Ago2 association and the resistance of miR-223 against RNaseA was observed during all trans retinoic acid (ATRA)-induced cell differentiation of promyelocytic HL60 cells. In conclusion, the association of miRNAs with Ago2 complexes, an event that is linked to cell functional status, plays a critical role in stabilizing the circulating miRNAs in cell-secreted MVs.