Bisphosphonates are the mainstay of therapy worldwide for osteoporosis. However, bisphosphonates still have their limitations. Our study implied miR-101-3p is a important regulator in bisphosphonates treated-osteoclasts. When miR-101-3p were down-regulated in bone marrow-derived macrophage-like cells (BMMs), the development of mature osteoclasts were promoted, and vice versa. However, alendronate can decrease the multinucleated cell whether miR-101-3p is knock-down or over-expression. TRAP activity assay also confirmed above results.Luciferase assay indicated that miR-101-3p is a negative regulator of Rap1b and western blot analysis displayed that protein expression level of Rap1b in BMMs transfected with OV-miR-101-3p was lower than in BMMs transfected with empty vector. And Rap1b overexpression can increase the TRAP-positive multinucleated cells, and vice versa. Furthermore, in vivo data showed that miR-101-3p could inhibit osteoclast differentiation in ovariectomized mice,while overexpressed Rap1b can block it. Our data demonstrate that miR-101-3p/Rap1b signal pathway played a key role in osteoclast differentiation after treatment with bisphosphonates.