The vitamin D levels in mothers affect the health status of both the mother and breastfeeding infant. Vitamin D deficient mothers' infants are prone to rickets. While tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been implicated in cellular growth/apoptosis, immune cell function and bone-resorbing osteoclast formation, the expression of TRAIL in human milk as a function of vitamin D status in mothers remains unknown. We hypothesized that vitamin D deficiency alters TRAIL protein levels in human breast milk and mammary epithelial cells. Milk from vitamin D deficient mothers showed high levels of TRAIL (α and β) proteins compared to milk from vitamin D replete women. Western blot analysis of total cell lysate obtained from normal human mammary epithelial (HME-1) cells treated with variable doses (0-20 nM) of vitamin D for 24 h demonstrated that low levels (0.5 to 5 nM) significantly increased the TRAIL α but no change in β expression. In contrast, vitamin D at 20 nM concentration suppressed the expression of both TRAIL α and β proteins. Consistently, vitamin D regulated TRAIL mRNA expression in HME-1 cells. Our results indicate that vitamin D status in mothers modulates TRAIL expression in breast milk, which may have implications for both mother and infant health.