The development of nonalcoholic fatty liver disease (NAFLD) is closely related to the fatty acid (FA) uptake. This study aimed to investigate the effect of Krüppel-like factor 9 (KLF9) on CD36 (typical fatty acid translocase), hepatocellular lipid metabolism as well as the development and progression of nonalcoholic fatty liver. High-fat diet-induced obese C57BL/6J mice and db/db mice were used to test the expression levels of Klf9 and Cd36 in the livers. The primary hepatocytes were isolated from C57BL/6J mice, treated with Ad-GFP, Ad-Klf9, Ad-shCtrl or Ad-shKlf9, and then incubated with oleic acid and palmitic acid for 24 h. Liver-specific knockout of Klf9 mice were established. The protein levels and relative mRNA levels were examined by Western blot and real-time PCR, respectively. Triglyceride content was determined by using an assay kit. Lipid content was determined by Oil Red O staining. The results showed that: (1) Klf9 expression levels were increased in the livers of high-fat diet-induced obese mice and db/db mice, compared to their respective control mice. (2) Adenovirus-mediated overexpression of Klf9 in primary hepatocytes increased Cd36 expression and cellular triglyceride contents. (3) In contrast, adenovirus-mediated knockdown of Klf9 expression in primary hepatocytes by Ad-shKlf9 decreased Cd36 expression and cellular triglyceride contents. (4) Finally, Klf9 deficiency decreased liver Cd36 expression and alleviated fatty liver phenotype of high-fat diet-induced obese mice. These results suggest that KLF9 can regulate hepatic lipid metabolism and development of NAFLD by promoting the expression of CD36.