The aim of this study was to evaluate the effect of rumen-protected niacin (RPN) supplementation during summer months on vaginal temperature, milk production, and milk composition of dairy cows. The study was conducted as switchback design constituted of 3 consecutive 3-week periods. Fifty lactating multiparous Holstein cows > 100 days in milk were randomly assigned to one of two treatments groups. Dietary treatments were: (1) control diet (CON; n=25), not supplemented with RPN and (2) supplemented diet (RPN; n=25), supplemented with RPN (15 g/cow daily). Both diets were fed for ad libitum intake 3X daily. Cows remained in the study for 9 weeks. Temperature humidity index (THI) was calculated with ambient temperature and relative humidity recorded throughout the study. Vaginal temperature was recorded during week 3, 6, and 9. Body weight (BW) and body condition score (BCS) were measured weekly. Milk production was recorded daily. Milk samples collected twice weekly in week 3, 6, and 9 were analyzed for fat, protein, lactose, urea nitrogen, and somatic cell count. Data were analyzed using the MIXED procedure of SAS (v9.4). Two statistical analyses were performed: (1) all experimental periods (mean THI=67) and (2) period two only (mean THI=72). Diets with similar chemical-nutritive composition were fed though the study. Ambient conditions were similar for both treatments but different across periods. Analysis one revealed no effect of RPN on vaginal temperature. Supplementation of RPN did not increased milk yield, whereas milk fat concentration was higher in RPN cows. Analysis two revealed lower (P=0.01) highest vaginal temperature by day (24 h period) and nighttime period (1900-0459 h) in RPN cows. Mean vaginal temperature at nighttime tended to be lower (P=0.07) for RPN as well. Milk fat concentration was greater in RPN than CON cows, but milk yield was similar across treatments. However, trends towards increased milk yield were observed in RPN cows during the period of higher temperatures and higher mean THI. In both analysis BW and BCS were unaffected by RPN supplementation. Supplementation of RPN increased milk fat concentration but had no effect on milk yield. However, in the period of higher ambient temperature and higher mean THI trends towards greater milk yield and lower vaginal temperatures were observed in RPN supplemented cows. (C) 2016 Elsevier B.V. All rights reserved.