Vitamin D3 upregulated protein 1 (VDUP1) is a stress-response gene that is upregulated by 1,25(OH)2D3 in tumor cells. The in vivo roles of VDUP1 were investigated by producing mice lacking VDUP1 (VDUP1−/− mice). VDUP1−/− mice showed minimal changes in the development of T and B cells, but there was a profound reduction in the numbers of natural killer (NK) cells. As well, these mice showed decreased NK activity. In the VDUP1−/− mice, the expression of CD122 was reduced, demonstrating that VDUP1 is required for CD122 expression and NK maturation. In addition, severe lymphoid hyperplasia in the small intestine was observed in VDUP1−/− mice. Taken together, these results suggest that VDUP1 is a critical factor for the development and function of NK cells in vivo.