Our recent study showed that miR-2861 promotes osteoblast differentiation by targeting histone deacetylase 5, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Here we identified another new microRNA (miRNA) (miR-3960) that played a regulatory role in osteoblast differentiation through a regulatory feedback loop with miR-2861. miR-3960 and miR-2861 were found clustered at the same loci. miR-3960 was transcribed during bone morphogenic protein 2 (BMP2)-induced osteogenesis of ST2 stromal cells. Overexpression of miR-3960 promoted BMP2-induced osteoblastogenesis. However, the inhibition of miR-3960 expression attenuated the osteoblastogenesis. Homeobox A2 (Hoxa2), a repressor of Runx2 expression, was confirmed to be a target of miR-3960. Electrophoretic mobility shift assay and chromatin immunoprecipitation experiments confirmed that Runx2 bound to the promoter of the miR-3960/miR-2861 cluster. Furthermore, overexpression of Runx2 induced miR-3960/miR-2861 transcription, and block of Runx2 expression attenuated BMP2-induced miR-3960/miR-2861 transcription. Here we report that miR-3960 and miR-2861, transcribed together from the same miRNA polycistron, both function in osteoblast differentiation through a novel Runx2/miR-3960/miR-2861 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast differentiation.