Mitochondria regulate various cellular processes. Though highly active anti-retrovirus therapy (HAART) is effective cure for HIV, the therapy also causes life-threatening clinical manifestations resulting from mitochondrial toxicity caused by nucleoside reverse transcriptase inhibitors (NRTIs) involving 3ꞌ-azido 3ꞌ-deoxythymidine (AZT), which are a key component of HAART. The mechanism underlying the mitochondrial toxicity of NRTIs, however, remains uncertain. An active metabolite of AZT (AZT triphosphate, AZT-TP) is responsible for AZT toxicity. Mitochondrial permeability transition pore (mPTP) is a nonspecific channel permeable to any molecules < 1.5 kDa that penetrates the mitochondrial inner and outer membranes by forming a complex with cyclophilin D (CypD) locating in the matrix, ATP/ADP translocator lying at the inner membrane, and the voltage dependent anion channel positioned at the outer membrane. The mPTP-opening causes mitochondrial dysfunction. In this study, we examined the role of CypD on AZT-induced mitochondrial dysfunction. CypD expression was inhibited by RNAi. In the control cells, cyclosporin A (CsA) which binds to CypD blocks the mPTP-opening caused by AZT and restores the mitochondrial function impaired by AZT. Knockdown of the CypD abolished the effects of CsA on the inhibition of mPTP-opening caused by AZT and made worse the mitochondrial function. These results suggest that CypD is prerequisite for the inhibition of mPTP-opening by CsA.