The aim of the present study was to investigate the effect of 1,25(OH)2D3/vitamin D receptor (VDR) and calcium on the expression levels of osteogenic factors in primary renal tubular epithelial cells (RTECs) using genetic hypercalciuric rats. The basal levels of osteogenic factors were detected in Sprague Dawley and genetic hypercalciuric rats. The gene and protein levels of bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2) and osterix were detected in the RTECs transduced with Lenti-VDR-sh and were incubated with calcium. Using the o-cresolphthalein complexone method, the calcium levels of the primary RTECs cultured with Lenti-VDR-sh and with 1,25(OH)2D3 were assessed. The basal levels of BMP2, Runx2 and Osterix in the cells were significantly higher in the genetic hypercalciuric rats compared with the control rats. VDR knockdown in the RTECs reduced the expression levels of BMP2, Runx2 and Osterix. The calcium depositions in the primary RTECs were also decreased following exposure to Lenti-VDR-sh, but increased following treatment with 1,25(OH)2D3. The expression levels of BMP2, Runx2 and Osterix were markedly increased in the cells incubated with calcium compared with the cells treated with normal saline and the untreated cells. These findings indicated that osteogenic factors, including BMP2, Runx2 and Osterix may be important in renal stone formation in idiopathic hypercalciuria. VDR may mediate the increased expression levels of BMP2, Runx2 and Osterix by positively regulating calcium levels in primary RTECs.