As a common bone disease in the elderly population, osteoporosis-related bone loss and bone structure deterioration represent a major public health problem. Therapeutic strategies targeting excessive osteoclast formation are frequently used for osteoporosis treatment; however, potential side effects have been recorded. Here, we have developed a novel therapeutic strategy using microdroplets (MDs) encapsulated with NFATc1-siRNA and investigated the role of bioactive MDs-NFATc1 biocompatibility in RAW 264.7 macrophages and human mesenchymal stem cells (hBMSCs), respectively. Its role in regulating osteoclast differentiation and formation was also investigated in vitro. We first fabricated MDs with spherical morphology along with a well-defined core-shell structure. The ultrasound-responsive study demonstrated time-dependent responsive structural changes following ultrasound stimulation. The internalization study into unstimulated macrophages, inflammatory macrophages, and hBMSCs indicated good delivery efficiency. Furthermore, the results from the MTT assay, the live/dead assay, and the cellular morphological analysis further indicated good biocompatibility of our bioactive MDs-NFATc1. Following MDs-NFATc1 treatment, the number of osteoclasts was greatly reduced, indicating their inhibitory effect on osteoclastogenesis and osteoclast formation. Subsequently, osteoporotic rats that underwent ovariectomy (OVX) were used for the in vivo studies. The rats treated with MDs-NFATc1 exhibited significant resistance to bone loss induced by OVX. In conclusion, our results demonstrate that MDs-NFATc1 could become an important regulator in osteoclast differentiation and functions, thus having potential applications in osteoclast-related bone diseases.