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.