Critically ill patients are frequently treated with empirical antibiotic therapy, including vancomycin and β-lactams. Recent evidence suggests an increased risk of acute kidney injury (AKI) in patients who received a combination of vancomycin and piperacillin-tazobactam (VPT) compared with patients who received vancomycin alone or vancomycin in combination with cefepime (VC) or meropenem (VM), but most studies were conducted predominately in the non-critically ill population. A retrospective cohort study that included 2,492 patients was conducted in the intensive care units of a large university hospital with the primary outcome being the development of any AKI. The rates of any AKI, as defined by the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines, were 39.3% for VPT patients, 24.2% for VC patients, and 23.5% for VM patients ( P < 0.0001 for both comparisons). Similarly, the incidences of stage 2 and stage 3 AKI were also significantly higher for VPT patients than for the patients in the other groups. The rates of stage 2 and stage 3 AKI, respectively, were 15% and 6.6% for VPT patients, 5.8% and 1.8% for VC patients, and 6.6% and 1.3% for VM patients ( P < 0.0001 for both comparisons). In multivariate analysis, the use of vancomycin in combination with piperacillin-tazobactam was found to be an independent predictor of AKI (odds ratio [OR], 2.161; 95% confidence interval [CI], 1.620 to 2.883). In conclusion, critically ill patients receiving the combination of VPT had the highest incidence of AKI compared to critically ill patients receiving either VC or VM.