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 and proteins 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, and suppressed the expression of the differentiation markers in osteoblasts. In addition, the expression levels of Kir2.1 proteins were up-regulated in murine embryonic metatarsals, depending on the progression of the endochondral ossification. These results suggest that Kir2.1 channels play essential roles in maintaining the bone homeostasis via modulating osteoblast differentiation.