The clinical significance of pre-transplant donor-specific antibodies (DSA), despite negative cytotoxicity and flow cytometry crossmatches (FCXMs), is unknown. We performed a retrospective cohort study of 60 living donor renal transplant recipients, all with pre-transplant cytotoxicity and T-cell and B-cell FCXMs that were negative. Twenty recipients had pre-transplant DSA detected by enzyme-linked immunosorbent assays (ELISA) and/or microbead methods. Forty contemporaneous DSA-negative controls were selected. In the DSA-positive group, after a median follow-up of 8.2 months (25-75% range, 5.4-22.8 months), patient survival was 100% and allograft survival was 95.0%. Acute humoral rejection (AHR) developed in four patients (20.0%). Three of the AHR episodes occurred within the first month post-transplant. Median serum creatinine at last follow-up was 1.3 mg/dL (25-75% range, 1.0-1.6 mg/dL), versus 1.1 mg/dL (25-75% range, 0.9-1.4 mg/dL) in the DSA-negative controls (p = 0.29). Only one of the 40 controls developed AHR (2.5%). Pre-transplant DSA was associated with a significantly increased incidence of AHR (p = 0.02 by log-rank test). In conclusion, despite negative pre-transplant cytotoxicity and FCXMs, renal transplant recipients with pre-transplant DSA detected by solid-phase methods may have an increased incidence of AHR and require close monitoring post-transplant.