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Heat stress (HS) has become a significant concern in livestock farming as it affects animal health, welfare, and productivity. The number of studies on the impact of HS on the uterine environment, however, is still limited. The objective of this study was to assess the effect of HS exposure on the proportion of polymorphonuclear neutrophils (PMN%) and the microbial growth density (MGD) in the postpartum uterus. Uterine cytobrush samples were taken from 52 dairy cows at days 7, 14, 28, 42 and 56 postpartum (dpp). Samples were used for cytology to determine the PMN% and for bacteriology to assess the MGD on the agar plates after aerobic cultivation. Ambient temperature and relative humidity were recorded at 30-min intervals with dataloggers installed in the barn and the temperature-humidity index (THI) was calculated. The accumulated HS exposure was calculated as the area under the curve between the THI and the threshold of 68 (Riemann sum). Positive correlations (r = 0.3–0.4, p < 0.05) were observed between short-term HS (48 h prior to sampling) and PMN% at 7, 14 and 42 dpp. The MGD correlated negatively with HS 12 h prior to sampling at 14 and 42 dpp (both r = −0.4, p < 0.05). Long-term HS (7, 14, 21 and 28 days prior to sampling) significantly correlated with PMN% and only at 42 dpp (r = 0.3–0.4, p < 0.05). Our results demonstrate that the effect of HS on PMN% and MGD depends on the duration and intensity of HS exposure and the day postpartum. Further research is needed to explore underlying mechanisms contributing to impaired uterine health and fertility under HS.