A growing body of evidence has revealed that microRNAs (miRNAs) play crucial roles in regulating osteoblasts and bone metabolism. However, the effects of miRNAs in osteoblast mechanotransduction remain to be defined. In this study, we investigated the regulatory effect of miR-33-5p in osteoblasts and tested its anti-osteopenia effect when delivered by an osteoblast-targeting delivery system in vivo. First, we demonstrated that miR-33-5p could promote the activity and mineralization of osteoblasts without influencing their proliferation in vitro. Then our data showed that supplementing miR-33-5p in osteoblasts by a targeted delivery system partially recovered the osteopenia induced by mechanical unloading at the biochemical, microstructural, and biomechanical levels. In summary, our findings demonstrate that miR-33-5p is a key factor in the occurrence and development of the osteopenia induced by mechanical unloading. In addition, targeted delivery of the mimics of miR-33-5p is a promising new strategy for the treatment of pathological osteopenia.