Objective: The objective was to evaluate the effects of UV-B irradiated vitamin D-enriched yeast supplementation on milk yield, milk composition, vitamin D in milk, milk fatty acids, blood chemistry, and 25(OH)D status in dairy cows. Methods: Six Thai Friesian cows (milk production, 11.2±2.0 kg/d; body weight, 415.0±20.0 kg; and days in milk, 90.0±6.0) were allocated to each treatment in a 3×3 Latin square design, with three treatments and three periods. Each period of the Latin square lasted 49 days consisting of 14 days for diet adaptation and 35 days for sample collection. Dairy cows were randomly assigned to one of three treatments: i) feeding a basal diet without yeast (CON); ii) basal diet + 5 g of live yeast (75 IU/head/d of vitamin D2; LY); and iii) basal diet + 5 g of UV-B irradiated vitamin D enriched yeast (150,000 IU/head/d of vitamin D2; VDY). Feed intake and milk production were recorded daily, milk sample collection occurred on days 14 and 35 of each collection period, and blood plasma was collected on days 0, 7, 14, 21, 28, and 35 of each collection period. Results: The results show that after a trial period of 14 and 35 days, the VDY group had significantly higher vitamin D content in milk than the LY and CON groups (376.41 vs 305.15, 302.14 ng/L and 413.46 vs 306.76, 301.12 ng/L, respectively). At days 7, 14, 21, 28, and 35 of the experiment, cows fed the VDY group had significantly higher 25(OH)D2 status in blood than the CON and LY groups (51.07 vs 47.16, 48.05 ng/mL; 54.96 vs 45.43, 46.91 ng/mL; 56.16 vs 46.87, 47.16 ng/mL; 60.67 vs 44.39, 46.17 ng/mL and 63.91 vs 45.88, 46.88 ng/mL), respectively. Conclusion: In conclusion, UV-B irradiated vitamin D-enriched yeast supplementation could improve vitamin D content in the milk and 25(OH)D status in dairy cows during the lactation period.