Objective To investigate the roles of osteopontin(OPN) and nuclear transcription factor in a rat model of chronic cyclosporine A (CsA) nephrotoxicity. Methods Male Sprague-Dawley rats maintained on a low salt diet were treated daily with vehicle (olive oil, 1 mL·kg^-1·d^-1, s.c.) and CsA (15 mL·kg^-1·d^-1, s.c.) for 4 weeks. Renal histopathology was estimated by trichrome staining (tubulointerstitial fibrosis) and immunohistochemistry (ED-1) to assess the degrees of renal tubulointerstitial lesions. In addition, OPN mRNA and protein expression and nuclear transcription factor (NF-κB and AP-1) were studied by northern blotting analysis, immunohistochemistry, electrophoretic mobility shift assay, and immunoblotting analysis. Results CsA-treated rats displayed significantly striped tubulointerstitial fibrosis ([38.9±3.3]%/5 mm² vs [0±0]%/5 mm², P<0.01) and increased ED-1-positive cells (89±9 vs 7±2, P<0.01). Compared with VH-treated rats, CsA-treated rats showed significantly upregulated OPN mRNA and protein expression in the proximal tubular cells, mainly localizing at areas of severe injured tissues. CsA-treated group also had significantly increased activities of NF-κB ([218±19]% vs [116±15]%, P<0.01) and AP-1 ([735±225]% vs [101±4]%, P<0.01), and significantly decreased expression of ([9±7]% vs [105±7]%, P<0.01). Correlation analysis revealed that upregulated OPN mRNA was positively correlated with tubulointerstitial fibrosis (r= 0.959, P<0.001) and activities of NF-κB and AP-1 (NF-κB: r=0.773, P<0.01; AP-1: r=0.619, P=0.01, respectively). Conclusion Our findings suggest that OPN, nuclear transcription factor NF-κB and AP-1 are involved in renal tubulointerstitial injury in chronic CsA nephrotoxicity.