Peroxisomes function together with mitochondria in a number of essential biochemical pathways, from bile acid synthesis to fatty acid oxidation1. Peroxisomes grow and divide from pre-existing organelles2, but can also emergede novoin the cell3. The physiological regulation ofde novoperoxisome biogenesis remains unclear, and it is thought that peroxisomes emerge from the endoplasmic reticulum in both mammalian and yeast cells4. However, in contrast to the yeast system5,6,7,8, a number of integral peroxisomal membrane proteins are imported into mitochondria in mammalian cells in the absence of peroxisomes, including Pex3, Pex12, Pex13, Pex14, Pex26, PMP34 and ALDP9,10,11,12,13,14,15. Overall, the mitochondrial localization of peroxisomal membrane proteins in mammalian cells has largely been considered a mis-targeting artefact in whichde novobiogenesis occurs exclusively from endoplasmic reticulum-targeted peroxins16. Here, in following the generation of new peroxisomes within human patient fibroblasts lacking peroxisomes, we show that the essential import receptors Pex3 and Pex14 target mitochondria, where they are selectively released into vesicular pre-peroxisomal structures. Maturation of pre-peroxisomes containing Pex3 and Pex14 requires fusion with endoplasmic reticulum-derived vesicles carrying Pex16, thereby providing full import competence. These findings demonstrate the hybrid nature of newly born peroxisomes, expanding their functional links to mitochondria.