Cyclophilin‐D (Cyp‐D) is a mitochondrial matrix peptidyl‐prolyl isomerase. Because cyclophilins can regulate nuclear gene expression, we examined whether Cyp‐D could regulate mitochondrial gene expression. We demonstrated in HEK 293T cells that transfected Cyp‐D interacts with mitochondrial transcription factors B1 and B2 (TFB2M) but not with mitochondrial transcription factor A. We also demonstrated that Cyp‐D interacts in vivo with TFB2M. Genetic silencing of Cyp‐D and pharmacologic inhibition of Cyp‐D markedly reduced mitochondrial transcription to 18 ± 5% (P < 0.05) and 24 ± 3% (P < 0.05) of respective controls. The level of interaction between Cyp‐D and TFB2M correlated with the level of nascent mitochondrial RNA intensity (r =0.896; P = 0.0156). Cyp‐D silencing down‐regulated mitochondrial transcripts initiated from the heavy strand promoter 2 [i.e., NADH dehydrogenase 1 (ND1) by 11‐fold, P < 0.005; cytochrome oxidase 1 (COX1) by 4‐fold, P < 0.001; and ATP synthase subunit 6 (ATP6) by 6.5‐fold, P < 0.005); but not NADH dehydrogenase 6 (ND6)], which is initiated from the light strand promoter. Cyp‐D silencing reduced mitochondrial membrane potential and cellular oxygen consumption (from 59 ± 5 to 34 ± 1 μmol oxygen/min/10⁶ cells, P< 0.001); the latter without a statistically significant reversal after uncoupling electron transport from ATP synthesis, consistent with down‐regulation of electron transport complexes. Accordingly, these studies provide novel evidence that Cyp‐D could play a key role in regulating mitochondrial gene expression.— Radhakrishnan, J., Bazarek, S., Chandran, B., Gazmuri, R. J. Cyclophilin‐D: a resident regulator of mitochondrial gene expression. FASEB J. 29, 2734‐2748 (2015). www.fasebj.org