Intracellular Ca²⁺ ([Ca²⁺]_i) signaling has a critical role in osteoblast proliferation and differentiation. Membrane hyperpolarization increases the driving force for Ca²⁺ signaling and promotes osteogenic differentiation in human mesenchymal stem cells. Therefore, K⁺ channels are key regulators of osteoblast differentiation, however, the functional role of K⁺ channels in osteoblast Ca²⁺ signaling remains unknown. In the present study, the contribution of K⁺ channels to [Ca²⁺]_i mobility 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 upregulated in the differentiated MC3T3-E1 cells. The application of ML133, a Kir2 inhibitor, significantly reduced the store-operated Ca²⁺ entry in differentiated MC3T3-E1 cells, and suppressed the expression of the differentiation markers in osteoblasts. The expression levels of Kir2.1 proteins were also upregulated in murine embryonic metatarsals, depending on the progression of the endochondral ossification. In addition, we showed that downregulation of miR-106b-5p augmented the expression levels of Kir2.1 in differentiated MC3T3-E1 cells. These results suggest that Kir2.1 channels play essential roles in maintaining the bone homeostasis via modulating osteoblast differentiation.