Background: Tysnd1 and PsLon are newly identified peroxisomal matrix serine proteases. Results: Tysnd1 inactivates its protease activity by self-conversion of the 60-kDa form to 15-and 45-kDa chains that are then degraded by PsLon. Tysnd1 regulates the peroxisomal fatty acid ␤-oxidation pathway via proteolytic processing of ␤-oxidation enzymes. Conclusion: Tysnd1 and PsLon cooperatively regulate fatty acid ␤-oxidation in peroxisomal matrix. Significance: The data shed light on how enzyme turnover is regulated inside peroxisomes.