Twenty-four multiparous high-producing dairy cows (40.0±1.4kg/d) were used in a factorial design to evaluate effects of 2 environments [thermoneutral (TN) and heat stress (HS)] and a dose range of dietary rumen-protected niacin (RPN; 0, 4, 8, or 12g/d) on body temperature, sweating rate, feed intake, water intake, production parameters, and blood niacin concentrations. Temperature-humidity index values during TN never exceeded 68 (stress threshold), whereas temperature-humidity index values during HS were above 68 for 24h/d. The HS environment increased hair coat and skin, rectal, and vaginal temperatures; respiration rate; skin and hair coat evaporative heat loss; and water intake and decreased DMI (3.5kg/d), milk yield (4.1kg/d), 4% fat-corrected milk (2.7kg/d), and milk protein yield (181.7g/d). Sweating rate increased during HS (12.7g/m(2) per h) compared with TN, but this increase was only 10% of that reported in summer-acclimated cattle. Niacin supplementation did not affect sweating rate, dry-matter intake, or milk yield in either environment. Rumen-protected niacin increased plasma and milk niacin concentrations in a linear manner. Heat stress reduced niacin concentration in whole blood (7.86 vs. 6.89μg/mL) but not in milk. Reduced blood niacin concentration was partially corrected by dietary RPN. An interaction existed between dietary RPN and environment; dietary RPN linearly increased water intake in both environments, but the increase was greater during HS conditions. Increasing dietary RPN did not influence skin temperatures. During TN, supplementing 12g/d of RPN increased hair coat (unshaved skin; 30.3 vs. 31.3°C at 1600h) but not shaved skin (32.8 vs. 32.9°C at 1600h) temperature when compared with 0g/d at all time points, whereas the maximum temperature (18°C) of the room was lower than skin temperature. These data suggest that dietary RPN increased water intake during both TN and HS and hair coat temperature during TN; however, core body temperature was unaffected. Thus, encapsulated niacin did not improve thermotolerance of winter-acclimated lactating dairy cows exposed to moderate thermal stress in Arizona.