This study was performed to evaluate the effects of maternal 25-hydroxycholecalciferol (25-OH-D₃) supplementation on sow performance, as well as the dynamic alterations of both the amino acid and fatty acid profiles in milk. On day 85 of gestation, twenty primiparous hybrid sows were allocated into two groups (10 sows/group) and fed a basal diet (3200 IU/kg vitamin D₃) containing either 0 or 50 μg/kg 25-OH-D₃ until weaning on d 21 of lactation. Milk was collected at 1, 3, 7, 14, and 21 d of lactation. The results showed that dietary 25-OH-D₃ supplementation notably decreased the score of tear stain at 101 d of gestation when compared to the Ctrl group (p = 0.030). No significant difference was found in terms of the gestation day, litter size, and litter weight at birth, whereas maternal 25-OH-D₃ intervention notably increased weaning weight and weight gain of the piglet (p < 0.05), while dietary 25-OH-D₃ supplementation contributed to a 16.4% body gain during lactation. The concentration of all amino acids in milk was higher in colostrum, following a dramatic drop. The rate of reduction for all amino acids was increased by dietary 25-OH-D₃ supplementation. The contents of saturated fatty acids and polyunsaturated fatty acids were increased and decreased linearly throughout lactation (both p < 0.05). Dietary 25-OH-D₃ supplementation initially suppressed both saturated and unsaturated fatty acid levels from d 1 to 7, while prompting a recovery of specific fatty acids from d 14 to 21 of lactation, particularly oleic acid, linoleic acid, and arachidonic acid. These findings indicate that maternal 25-OH-D₃ supplementation alters the pattern of milk fatty acid and amino acid composition, which may be associated with the observed improvement in piglet outcomes.