Updated project metadata. Peroxisomes are dynamic organelles with vital functions in cellular metabolism and dysfunction of peroxisomes is associated with human diseases. To fulfill their multiple roles, peroxisomes rely on import of nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p-2D) show a decreased peroxisomal import of GFP-SKL. We show that the binding affinity between PTS1-protein and Pex5p-2D is reduced. An in vivo analysis of the effect of the phosphomimicking mutants on all known PTS1-proteins revealed that import of most, but not all, cargo proteins is affected. The physiological effect of the phosphomimetic mutations correlate with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes.