Vitamin D (VD) plays important roles in bone development indirectly via control calcium absorption in the intestine and reabsorption in the kidney. However, several in vitro studies showed that VD can directly suppress the cell proliferation of mouse osteoblasts. The intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1 regulates intracellular Ca²⁺ signaling pathways and is associated with cell proliferation in various types of cells. In the present study, treatment with VDR agonists for 48 h markedly decreased the expression levels of K_Ca3.1 transcripts and proteins in mouse preosteoblast MC3T3-E1 cells, resulting in the significant inhibition of Ca²⁺ rises induced by DCEBIO, a specific K_Ca3.1 activator. Treatments with VDR agonists also significantly decreased the expression of transcriptional regulators of K_Ca3.1 such as histone deacetylase 2 (HDAC2) and Fra-1 composed of activation protein 1. In addition, the siRNA-mediated blockade of VDR significantly rescued the decreased expression of K_Ca3.1, HDAC2, and Fra-1 protein in MC3T3-E1 cells treated with VDR agonists. Our results suggest that K_Ca3.1 is a new downstream target of VDR signaling and the down-regulation of K_Ca3.1 contributes, at least partly, to the antiproliferative effect of VDR agonists in mouse pre-osteoblasts.