Store-operated Ca2+ entry (SOCE) plays critical roles in intracellular Ca2+ ([Ca2+]i) homeostasis. Recent studies have shown that SOCE is essential for osteoblastic differentiation. In non-excitable cells, K+ channels are key regulators of SOCE-mediated Ca2+ signaling and control cell proliferation, differentiation, and migration, however, the functional role of K+ channels in osteoblast Ca2+ signaling remains unknown. In the present study, the contribution of K+ channels to the SOCE activity in the osteoblastic cell line MC3T3-E1, established from mouse calvaria was investigated. We found that the expression levels of inward rectifier K+ channel Kir2.1 transcripts were up-regulated in the differentiated MC3T3-E1 cells. The application of ML133, a Kir2 inhibitor, significantly reduced the SOCE-mediated [Ca2+]i elevation in differentiated MC3T3-E1 cells, but not in immature MC3T3-E1 cells. In addition, the treatment with ML133 suppressed the expression of the differentiation markers in osteoblasts, and attenuated the endochondral ossification in murine embryonic metatarsals. These results suggest that Kir2.1 channels play essential roles in maintaining the bone homeostasis via modulating osteoblast differentiation.