<p>Human cytoplasmic leucyl-tRNA synthetase (LARS) is known to catalyze the ligation of leucine to tRNA^Leu during protein biosynthesis. However, LARS also acts as a nutrient sensor in a non-canonical activity to regulate cell growth. In this investigation, LARS was determined to be expressed at high levels in human liver cancer, which correlated with poor clinical outcomes in patients. Knockdown of LARS in HepG2 liver cancer cells suppressed cell proliferation and growth, yet promoted cell migration, without affecting global protein translation. Using data from RNA sequencing, differentially expressed genes in response to LARS knockdown were significantly clustered in the cellular senescence pathway. Elevated p21 and p16 expression with increased senescence-associated β-galactosidase activity was observed in LARS knockdown HepG2 cells. In addition, autophagy with increasing autophagic flux was triggered in LARS knockdown HepG2 cells. Furthermore, the depletion of LARS induced oxidative stress with increased production of reactive oxygen species and decreased mitochondrial membrane potential. Importantly, re-expression of shRNA-resistant LARS largely rescued the senescence, autophagy, and oxidative stress caused by LARS knockdown, confirming the specificity of these effects. These findings suggest that LARS plays a role in regulating liver cancer cell proliferation via multiple cellular responses and may serve as a promising therapeutic target in liver cancer.</p>