Anti-endothelial cell antibodies (AECAs) are thought to be involved in the development of renal allograft rejection. To explore this further, we determine whether AECAs play a role both in predicting the incidence of allograft rejection and long-term outcomes by analysis of serum samples from 226 renal allograft recipients for AECAs pre- and post-transplant. Surprisingly, the presence of pre-existing AECAs was not associated with either an increased risk of rejection or a detrimental impact on recipient/graft survival. Subsequent de novo AECAs, however, were associated with a significantly increased risk of early acute rejection. Moreover, these rejections tended to be more severe with a significantly increased incidence of both steroid-resistant and multiple episodes of acute rejection. The acute rejections associated with de novo AECAs did not correlate with C4d deposition at the time of renal biopsy, but did demonstrate an association with the presence of glomerulitis and peritubular capillary inflammation. Significantly more patients with de novo AECAs developed graft dysfunction. Thus, our prospective study suggests the emergence of de novo AECAs is associated with transplant rejection that may lead to allograft dysfunction.