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ABSTRACT: This study evaluated the effect of tannin extract from Acacia mearnsii on intravaginal temperature, production and milk composition of Holstein cows in a subtropical environment. The study period was 29 days and 20 multiparous Holstein cows were selected with an average milk production of 50 ± 6 kg/day and 147 ± 83 days in milk. The cows were separated into two treatments: Control (n = 10) did not receive dietary supplementation. Tannin (n = 10) received Condensed Tannin (Tanac S.A., Montenegro, RS, Brazil) at a dose of 0.19% dry matter (40 g/cow/day). Animals were monitored for milk production and composition as well as intravaginal temperature. The environmental temperature and humidity index (THI) were monitored. The mean THI was 72.39 ± 0.69. The mean intravaginal temperature was 39.17 ± 0.14 °C in the Tannin treatment and 39.03 ± 0.14°C in the Control treatment (P > 0.05). The animals in the Tannin treatment exhibited an intravaginal temperature of 39.2 °C at THI 69.38, and the Control treatment exhibited the same temperature at THI 70.16 (receiver operating characteristic (ROC); P < 0.01). Milk production was higher in the Tannin treatment than in the Control treatment (51.38 ± 0.90 kg/day and 49.94 ± 1.05 kg/day, respectively; P < 0.01). The protein content in milk was higher in the Tannin treatment than in the Control treatment (3.20 ± 0.01% and 3.04 ± 0.01%; respectively; P < 0.01). The results of this study suggested that tannin extract from Acacia mearnsii can attenuate heat stress and its negative effects on animal production.