The dysregulation of hepatic gluconeogenesis is a major factor in the pathogenesis of type 2 diabetes mellitus (T2DM). Hepatic gluconeogenesis is known to be tightly regulated at the transcription/expression level. The aim of this study was to evaluate the role of the E2F8 transcription factor in glucose metabolism. Here, we found that hepatic expression levels of E2F8 were increased in db/db and high-fat-diet-induced obese mice. Adenovirus-mediated overexpression of E2F8 in primary mouse hepatocytes upregulated expression of gluconeogenic genes, including those for PGC-1α, PEPCK, and G6Pase, subsequently increasing cellular glucose output. We demonstrated that E2F8 overexpression impairs insulin sensitivity in vitro. Furthermore, knockdown of E2F8 expression increased insulin sensitivity in primary hepatocytes. In summary, these findings indicated that E2F8 is involved in gluconeogenesis and insulin resistance and may represent a new therapeutic target in T2DM prevention.