Peroxisomes are metabolic organelles essential for human health. Defects in peroxisomal biogenesis proteins (peroxins/PEXs) cause devastating disease. PEX7 binds cargo proteins containing a type 2 peroxisomal targeting sequence (PTS2) to enable import of PTS2 proteins from the cytosol into peroxisomes. However, many aspects of PTS2-protein import remain enigmatic. Utilizing in vitro assays, yeast, and human cells, we show that PEX39, a previously uncharacterized protein, is a cytosolic peroxin that facilitates PTS2-protein import by binding PEX7 and stabilizing its interaction with PTS2 cargo. PEX39 and the N-terminus of PEX13, a peroxisomal membrane translocon protein, both possess a KPWE motif necessary for PEX7 binding. Sequential binding of PEX7 to this motif in PEX39 and PEX13 provides a novel paradigm for how PTS2 cargo engage the translocation machinery. Collectively, our work uncovers an ancient and functionally important relationship among PEX39, PEX7, and PEX13, offering insights that will advance our understanding of peroxisomal disease.