Background/objectives: CYP2A6 (CYP2A5 in mice) is mainly expressed in the liver. Hepatic CYP2A6 expression is increased in patients with non-alcoholic fatty liver disease (NAFLD). In mice, hepatic CYP2A5 is induced by high fat diet (HFD) feeding. Hepatic CYP2A5 is also increased in monosodium glutamate-induced obese mice. NAFLD is associated with obesity. In this study, we examined whether obesity is related to CYP2A6. Subjects/methods: Obesity genetic association study: The SAGE is a comprehensive genome-wide association study (GWAS) with case subjects having a lifetime history of alcohol dependence and control subjects never addicted to alcohol. We used 1030 control individuals with self-reported height and weight. A total of 12 single nucleotide polymorphisms (SNP) within the CYP2A6 gene were available. Obesity was determined as a BMI ≥30: 30–34.9 (Class I obesity) and ≥35 (Class II and III obesity). Animal experiment study: CYP2A5 knockout (cyp2a5 −/− ) mice and wild type (cyp2a5 +/+ ) mice were fed HFD for 14 weeks. Body weight was measured weekly. After an overnight fast, the mice were sacrificed. Liver and blood were collected for biochemical assays. Results: Single marker analysis showed that three SNPs (rs8192729, rs7256108, and rs7255443) were associated with class I obesity (p < 0.05). The most significant SNP for obesity was rs8192729 (odds ratio (OR) = 1.94, 95% confidence intervals = 1.21–3.10, p = 0.00582). After HFD feeding, body weight was increased in cyp2a5 −/− mice to a greater extent than in cyp2a5 +/+ mice, and fatty liver was more pronounced in cyp2a5 −/− mice than in cyp2a5 +/+ mice. PPARα deficiency in cyp2a5 −/− mice developed more severe fatty liver, but body weight was not increased significantly. Conclusion: CYP2A6 is associated with human obesity; CYP2A5 protects against obesity and NAFLD in mice. PPARα contributes to the CYP2A5 protective effects on fatty liver but it opposes to the protective effects on obesity.