Leucine (Leu) is a multifunctional essential amino acid that plays crucial role in various cellular processes. However, the integral effect of Leu on the hepatic proteome remains largely unknown. Here, we for the first time applied an isobaric tags for relative and absolute quantification (iTRAQ)-based comparative proteomics strategy to investigate the proteome alteration induced by Leu deprivation in human hepatocellular carcinoma (HepG2) cells. A total of 4,111 proteins were quantified; 43 proteins were further identified as differentially expressed proteins between the normal and Leu deprivation groups. Bioinformatics analysis showed that the differentially expressed proteins were involved in various metabolic processes, including amino acid and lipid metabolism, as well as degradation of ethanol. Interestingly, several proteins involved in the fatty acid β-oxidation pathway, including ACSL1, ACADS, and ACOX1, were up-regulated by Leu deprivation. In addition, Leu deprivation led to the reduction of cellular triglycerides in HepG2 cells. These results reveal that the fatty acid β-oxidation pathway is activated by Leu deprivation in HepG2 cells, and provide new insights into the regulatory function of Leu in multiple cellular processes, especially fatty acid metabolism.