Cyclophilin D (CypD) is an important component in mitochondrial‐dependent tubular cell death in acute kidney injury. However, it is not known whether CypD contributes to tubular cell damage in chronic interstitial fibrosis. We investigated this question in the unilateral ureter obstruction (UUO) model of renal interstitial fibrosis. Groups of CypD^−/− and wild type (WT) mice were killed 7 or 12 days after UUO surgery. The significant tubular cell apoptosis seen in WT UUO was significantly reduced in CypD^−/− UUO based on TUNEL and cleaved caspase 3 staining. Other markers of tubular cell damage; loss of E‐cadherin and AQP1 expression, were also reduced in the CypD^−/− UUO kidney. This reduced tubular damage was associated with less inflammation and a partial protection against loss of peritubular capillaries. The prominent accumulation of α‐SMA+ myofibroblasts and interstitial collagen deposition seen in WT UUO was significantly reduced in CypD^−/− UUO on day 12, but not day 7. Activation of several pro‐fibrotic signalling pathways (p38 MAPK, JNK and Smad3) was unaltered in CypD^−/− UUO, arguing that CypD acts independently to promote renal fibrosis. CypD deletion in cultured tubular cells attenuated oxidative stress‐induced pro‐inflammatory, pro‐fibrotic and apoptotic responses; however, responses to angiotensin II and LPS were unaffected. In contrast, CypD deletion in cultured renal fibroblasts did not affect PDGF‐induced proliferation or TGF‐β1‐induced collagen I expression, suggesting no direct role of CypD in the fibroblast response. In conclusion, we have identified a role for CypD in chronic tubular cell damage and in the development of renal interstitial fibrosis.