<i>Background:</i> Ischemia-reperfusion (I/R) is present at various degrees in kidney transplants. I/R plays a major role in early function and long-term survival of renal allograft. The purpose of our study was to determine if immunosuppressants modulate I/R in a model that separates I/R from all immune responses. <i>Methods:</i> Sprague-Dawley rats with monolateral renal I/R received daily cyclosporine (A), tacrolimus (B), sirolimus (C) or saline (D). Sham-operated rats received saline (E). After 30 days, glomerular filtration rate for each kidney was measured by inulin clearance. Kidney injury was examined, and TGF-β, fibronectin and metalloproteases were evaluated by real-time PCR, Western blot and zymography. <i>Results:</i> Sirolimus, but not cyclosporine and tacrolimus, prevented a glomerular filtration rate decrease in I/R kidneys (403 ± 303 vs. 1,006 ± 484 µl/min, p < 0.05; 126 ± 170 vs. 567 ± 374 µl/min, p < 0.05; 633 ± 293 vs. 786 ± 255; A, B and C group, respectively, I/R vs. contralateral kidneys). Sirolimus reduced ED-1+ cell infiltrate, interstitial fibrosis and intimal thickening of small vessels observed in I/R kidneys of controls and calcineurin inhibitor-treated rats. Tacrolimus and cyclosporine increased fibronectin and TGF-β expression and matrix deposition. Only sirolimus increased metalloprotease activity. <i>Conclusions:</i> Sirolimus but not calcineurin inhibitors prevented I/R-induced kidney injury.