The clinical significance of non-HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large-scale studies incorporating analysis of multiple non-HLA antibodies simultaneously. We developed a multiplex non-HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non-HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP-dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased-donor kidney (N=3276) but not in recipients of a living-donor kidney (N=1496). At 10 years after deceased-donor transplantation, recipients with anti-ARHGDIB antibodies (94/3276=2.9%) had a 13% lower death-censored covariate-adjusted graft survival compared to the anti-ARHGDIB-negative (3182/3276=97.1%) population (Hazard ratio 1.82; 95% confidence interval, 1.32-2.53; p=0.0003). These antibodies occur independently from DSA or other non-HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non-HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti-ARHGDIB antibodies in all patients awaiting deceased-donor transplantation. This article is protected by copyright. All rights reserved.