Selenoprotein F (Selenof) is an endoplasmic reticulum (ER)-resident protein. It may be functionally linked to glycoprotein folding in the ER but the detail function is not fully understood. To study the function of Selenof, we used CRISPR/Cas9 to generate Selenof knockout mice and performed proteomic analysis of hepatic proteins by iTRAQ. Collectively, 83 differently expressed proteins (DEPs) were identified in the liver of Selenof knockout mice. The changes of mRNA and protein levels of 6 selected DEPs, Fatty acid synthase, ATP-citrate synthase, Glutathione S-transferase P 1, Transformer-2 protein homolog beta, Pyruvate kinase, Metallothionein-2, were further verified by quantitative real-time PCR or Western blot. The roles of 83 DEPs are mainly related to metabolism and cancer. Consistently, the levels of NADPH and ATP, two molecules closely related with energy metabolism, are significantly changed in the livers of Selenof knockout mice. SIGNIFICANCE: Our study identified potential biological pathways and proteins related to Selenof deletion. These findings will provide possible proteins/pathways related to Selenof and help to understand the function of Selenof and its relationship with certain diseases.