Treatment of osteoporosis is still a challenge in clinic, which leads to an increasing social burden as the aging of population. Exosomes originated from human adipose-derived stem cells (hASCs) hold promise to promote osteogenic differentiation, thus may ameliorate osteoporosis. The main purpose of this study was to investigate the novel usage of hASC-derived exosomes in the treatment of osteoporosis and their underlying mechanism. Two types of exosomes, i.e., exosomes derived from hASCs cultured in proliferation medium (P-Exos) and osteogenic induction medium (O-Exos), were obtained. As compared with P-Exos, O-Exos could promote the osteogenic differentiation of mouse bone marrow-derived stem cells (mBMSCs) from osteoporotic mice in vitro and ameliorated osteoporosis in vivo. Then, microRNA (miRNA)-335-3p was identified to be the key differentially expressed microRNA between the two exosomes by small RNA sequencing, gene overexpression and knock-down, qRT-PCR, and dual-luciferase reporter assay, and Aplnr was confirmed to be the potential target gene of miRNA-335-3p. In addition, miR-335-3p inhibitor-optimized O-Exos were established by transfection of miR-335-3p inhibitor, which significantly enhanced the osteogenic differentiation of mBMSCs in vitro, and bone density and number of trabecular bones in vivo compared with unoptimized O-Exos. Our results indicated that the ASC-exosome-based therapy brings new possibilities for osteoporosis treatment. Besides, engineered exosomes based on transfection of miRNA are a promising strategy to optimize the therapeutic effect of exosomes on osteoporosis.