Peroxisome biogenesis requires up-to-now unidentified mitochondrial proteins. We show that the outer mitochondrial membrane (OMM) associated E3 Ub ligase MARCH5 is critical for early steps of generating mitochondria-derived peroxisomes. MARCH5 deficiency leads to an accumulation of immature peroxisomes, as well as lower expression of several peroxisomal proteins. When peroxisome biogenesis is induced through exposure of cells to fatty acids, MARCH5 is found in newly formed peroxisomes; however, peroxisome biogenesis is lost in MARCH5 deficient cells. Additionally, cells deficient in PEX3 and PEX14 peroxisome biogenesis factors exhibit impaired ability to generate peroxisomes and fail to indicate peroxisome biogenesis rescue if MARCH5 is simultaneously knocked out. WT MARCH5 re-expression pre-peroxisome formation in PEX14/MARCH5 DKO cells. PEX3 re-expression only reintroduces peroxisome formation in PEX3 KO, but not PEX3/MARCH5 DKO cells. Furthermore, our data also show reduced cellular ATP in a mitochondria-reliant energy generation model in MARCH5 KO cells. Our data suggest MARCH5 is essential for mitochondria dependent peroxisome biogenesis and may play a direct role in cellular energetics when cells are reliant on β-oxidation for energy generation.