Temperature increases due to climate change induce heat stress in livestock, resulting in economic losses for farmers. This study investigated the physiological responses of Hanwoo cattle to heat stress, and the effects of heat stress on the vaginal microbiome. Four cows (mean weight: 380.3 ± 37.5 kg, parity: 2.5 ± 1.0) were allocated to two groups: heat stress (temperature-humidity index (THI) 86: 33 °C, 70% humidity) and no heat stress (THI 67: 20 °C, 60% humidity). The cows were exposed to these conditions for 15 days, and the study was repeated twice with the same cohort of animals, ensuring that each cow was exposed to both thermal conditions in a crossover manner. The study revealed that feed intake was lower, while water intake, rumen temperature, rectal temperature, and respiration rate were higher in THI 86 than in THI 67 cows. The blood cortisol, glucose, and cholesterol concentrations, and the inverse Simpson index of the vaginal microbiome, were lower in THI 86 cows, while certain microorganisms (for example, Tenericutes) were more abundant in THI 86 than in THI 67 cows. The increase in blood oestradiol concentration during oestrus was 2.8× lower, and the duration of oestrus was shorter by 41 hours in the THI 86 than in the THI 67 cows. Blood progesterone concentrations increased after ovulation in THI 67 cows and during oestrus in THI 86 cows. In THI 86 cows, the increase in blood luteinising hormone secretion and ovulation were delayed by 10 hours and 11 hours, respectively. We concluded that heat stress raises the body temperature of Hanwoo cows, causes changes in endocrine hormones and metabolic physiology, reduces the microbial diversity in the vagina, and negatively affects the oestrus cycle.