Pharmacogenomics is a rapidly expanding field that delves into how an individual’s genome impacts their drug response. With the growing number of drug-related cases and the emergence of precision medicine, incorporating pharmacogenomics into forensic and clinical practice is becoming increasingly crucial.Cytochrome P450 2B6 is a drug-metabolizing enzyme that plays a significant role in the metabolism of various drugs, including cyclophosphamide, efavirenz, methadone, and ecstasy. A deficiency in CYP2B6 can make an individual a poor metabolizer, which can lead to overdose or even death. Therefore, a better understanding of CYP2B6 regulation would be valuable to forensic toxicologists and clinical practitioners. This study focuses on CYP2B6 regulation by ATF5, a basic leucine zipper transcription factor that transactivates CYP2B6 in liver cells. Studies have shown that a cell-penetrating dominant-negative ATF5 (CP-DN-ATF5) peptide can interfere with ATF5-mediated transactivation of anti-apoptotic proteins, eventually triggering tumor cell apoptosis. Our study aimed to determine whether CP-DN-ATF5 could also hinder the ATF5-mediated transactivation of CYP2B6, leading to CYP2B6 downregulation and impacting drug metabolism. CP-DN-ATF5 peptide treatment to two types of glioblastoma cell lines, GBM5 and LN229, followed by western blot analyses, revealed that CP-DN-ATF5 peptide effectively downregulates CYP2B6 protein level.