This study is designed to evaluate the effect of 25-hydroxyvitamin D3 (25(OH)D3) and divalent calcium ion (Ca²⁺) supplementation in sows’ diets on trace elements in blood and milk to assess whether it is beneficial to pig growth or not. A total of 40 sows of 7 to 8 gestational age with good health and nutritional condition were divided into four groups: control group, low calcium group, 25(OH)D3 group and low calcium with 25(OH)D3 group. The blood and milk from sows and neonatal piglets’ blood were collected on the day of parturition. Changes in the content of Cr, Mn, Cu, As, Se and Cd were analyzed. The results showed that there was no significant change in the content of Cr, Mn, Se and Cd in sows’ blood by changing calcium content or adding 25(OH)D3 alone, while adding 25(OH)D3 alone can significantly reduce the content of copper in sows' blood. Under the combined action of low calcium and 25(OH)D3, the Cr, Mn and Cd content in piglets’ blood were significantly lower than that of control group (P<0.05), and the content of copper was significantly increased compared to control group (P<0.01). Low calcium group and 25(OH)D3 group significantly increase the content of Cr, Cu and Se in sows’ milk compared to control group (P<0.05), but has no effect on the content of arsenic. Altogether, our results suggest that addition of 25(OH)D3 to sows’ diet is beneficial to maintain the balance of trace elements in sows and their fetuses, so 25(OH)D3 may be helpful for the health of mother and normal development of fetus during pregnancy.