Calcitriol, also known as 1,25-dihydroxyvitamin D3 (1,25(OH) 2VD 3), is a biologically active form of vitamin D and has a wide range of anticancer activity against various cancer cell lines. However, the mechanism of calcitriol remains to be further studied. In this study, the biological effect and epigenetic regulation of calcitriol on kidney cancer cells were investigated. Calcitriol can significantly inhibit cell proliferation of kidney cancer cell lines 786-O ( P<0.05). Calcitriol also induced cell apoptosis and senescence of 786-O and ACHN ( P<0.05). Calcitriol can increase the expression of histone demethylase JMJD3 and cell senescence marker p16INK4A ( P<0.05). Knockdown of JMJD3 decreased p16INK4A upregulation after calcitriol treatment ( P<0.05), and also reduced calcitriol-induced cell senescence ( P<0.05). This study reveals a new mechanism of anticancer activity of calcitriol by showing that histone demethylase JMJD3 induced by calcitriol increases p16INK4A expression and cell senescence. Therefore, these results provide new strategy for treatment and prevention of kidney cancer.