Peroxisomes are integral metabolic organelles involved in both catabolic and anabolic processes in humans, with defects linked to diseases. The functions of peroxisomes are regulated at transcriptional, translational, and posttranslational levels. In this study, we employed the CRISPR/Cas9-based screening of a ubiquitin ligase library to identify regulators of human peroxisomes. We discovered that ZBTB17 (MIZ1) plays a role in regulating the import of proteins into peroxisomes. Independent of its ubiquitin ligase activity, ZBTB17/MIZ1 operates as a transcription factor to modulate the expression of key importer PEX13, influencing the localization of peroxisomal enzymes. Furthermore, metabolomic profiling reveals that knockdown of ZBTB17 or PEX13 results in similar metabolic alterations, with downregulated purine synthesis. Collectively, we identify ZBTB17 as a key regulator of peroxisomal protein import, thereby affecting peroxisomal function and nucleotide metabolism. Our findings provide insights into the multifaceted regulation of peroxisomes in complex human cells and shed light on the molecular mechanisms underlying ZBTB17's role as a transcriptional regulator.