The combination of hyperthermia, dehydration, and strenuous exercise can result in severe reductions in kidney function, potentially leading to acute kidney injury (AKI). We sought to determine whether six days of heat acclimation (HA) mitigates the rise in clinical biomarkers of AKI during strenuous exercise in the heat. Twenty men completed two consecutive 2 h bouts of high-intensity exercise in either hot (n = 12, 40 °C, 40% relative humidity) or mild (n = 8, 24 °C, 21% relative humidity) environments before (PreHA) and after (PostHA) 4 days of 90–120 min of exercise per day in a hot or mild environment. Increased clinical biomarkers of AKI (CLINICAL) was defined as a serum creatinine increase ≥0.3 mg·dL−1 or estimated glomerular filtration rate (eGFR) reduction >25%. Creatinine similarly increased in the hot environment PreHA (0.35 ± 0.23 mg·dL−1) and PostHA (0.39 ± 0.20 mg·dL−1), with greater increases than the mild environment at both time points (0.11 ± 0.07 mg·dL−1, 0.08 ± 0.06 mg·dL−1, p ≤ 0.001), respectively. CLINICAL occurred in the hot environment PreHA (n = 9, 75%), with fewer participants with CLINICAL PostHA (n = 7, 58%, p = 0.007), and no participants in the mild environment with CLINICAL at either time point. Percent change in plasma volume was predictive of changes in serum creatinine PostHA and percent changes in eGFR both PreHA and PostHA. HA did not mitigate reductions in eGFR nor increases in serum creatinine during high-intensity exercise in the heat, although the number of participants with CLINICAL was reduced PostHA.