Previous reports have revealed that Prospero‐related homeobox 1 (Prox1) is required for the migration and differentiation of hepatoblasts during embryonic liver formation. However, the role of Prox1 in adults remains to be elucidated. We created liver‐specific Prox1 knockout mice to verify the role of Prox1 in adult hepatocytes. The mutant mice exhibit hepatic injury and a nonobese, insulin‐resistant diabetic phenotype in vivo. Hepatocyte injury is observed predominantly in the perivenous region and is characterized by the formation of vacuoles and emergence of round‐shaped mitochondria, suggesting that the effect of Prox1 on the maintenance of adult hepatocytes is region dependent. Furthermore, glycolysis is suppressed, and both oxidative phosphorylation and autophagy are upregulated in the livers of Prox1 knockout mice, indicating that Prox1 has a role in regulating energy homeostasis in hepatocytes.