The maturity of osteoblasts by proliferation and differentiation in preosteoblasts is essential for maintaining bone homeostasis. The beneficial effects of vitamin D on bone homeostasis in mammals have been demonstrated experimentally and clinically. However, the direct actions of vitamin D on preosteoblasts remain to be fully elucidated. In this study, we found that the functional activity of intermediate-conductance Ca²⁺-activated K⁺ channels (K_Ca3.1) positively regulated cell proliferation in MC3T3-E1 cells derived from mouse preosteoblasts by enhancing intracellular Ca²⁺ signaling. We examined the effects of treatment with vitamin D receptor (VDR) agonist on the expression and activity of K_Ca3.1 by real-time PCR examination, Western blotting, Ca²⁺ imaging, and patch clamp analyses in mouse MC3T3-E1 cells. Following the downregulation of K_Ca3.1 transcriptional modulators such as Fra-1 and HDAC2, K_Ca3.1 activity was suppressed in MC3T3-E1 cells treated with VDR agonists. Furthermore, application of the K_Ca3.1 activator DCEBIO attenuated the VDR agonist-evoked suppression of cell proliferation rate. These findings suggest that a decrease in K_Ca3.1 activity is involved in the suppression of cell proliferation rate in VDR agonist-treated preosteoblasts. Therefore, K_Ca3.1 plays an important role in bone formation by promoting osteoblastic proliferation under physiological conditions.