Background/Aims: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide and is driven by metabolic reprogramming that supports tumor growth and progression. A common missense genetic variant (rs2642438, p.A165T) in mitochondrial amidoxime reducing component 1 (MTARC1), identified as protective against liver disease, has been recently associated with lower prevalence of steatosis, cirrhosis, and HCC. However, the mechanistic role of MTARC1 in HCC is unclear. Therefore, we sought to decipher the role of MTARC1 in HCC. Methods: We investigated the role of MTARC1 in HCC by performing siRNA-mediated knockdown across human immortalized HCC cell lines (Hep3B2, HuH7, HepG2 and HepaRG) homozygous for the risk allele (p.A165) and by generating stable CRISPR-Cas9 knockout (KO) models. Next, we assessed the effect of MTARC1 loss on cell proliferation, migration, lipid metabolism, and fatty acid oxidation in vitro, as well as tumor aggressiveness in a subcutaneous xenograft mouse model. Additionally, we performed global proteomics in both in vitro and xenograft models. Results: Transient knockdown of MTARC1 p.A165 reduced proliferation in HCC cell lines. CRISPR-Cas9-mediated stable MTARC1 p.A165 KO in Hep3B2 cells led to decreased neutral lipid intracellular accumulation, enhanced β-oxidation and reduced cell migration. An MTARC1 KO xenograft model had reduced tumor volume. Proteomic analyses of both in vitro HCC cells and xenograft tumors revealed inhibition of oncogenic pathways and activation of anti-proliferative proteins. Conclusions Downregulation of MTARC1 p.A165 inhibits lipid accumulation, dampens tumor-promoting pathways and restricts tumor growth, highlighting MTARC1 as a promising therapeutic target for HCC.