Members of the mammalian long-chain acyl-CoA synthetase (ACSL) family are key enzymes for cellular fatty acid metabolism that catalyze the initial step in activation of long-chain fatty acids. However, the specificity of individual isoforms of ACSL to the lipid metabolic process is not well studied. In addition, the regulation of expression of individual ACSL isoforms under hyperlipidemic conditions is largely unknown. We cloned the hamster ACSL3 cDNA coding region and generated specific antibodies recognizing the ACSL3 protein. We next observed the changes in ACSL3 mRNA and protein expression in hamsters fed a standard chow diet or a high fat and high cholesterol (HFHC) diet. HFHC feeding significantly increased ACSL3 mRNA and protein expression in liver and to a lesser extent in muscle but not in adipose, brain, heart, or testis. Additionally, ACSL3 mRNA abundance was differentially regulated by the nutritional status in different tissues with liver, muscle, and adipose being the most sensitive tissues. Importantly, the hepatic ACSL3 mRNA expression pattern in response to fasting and refeeding in hyperlipidemic hamsters differed from that observed in normal chow-fed hamsters. Together, these results provide the first in vivo evidence of altered regulation of hepatic ACSL3 expression under hyperlipidemic conditions and suggest important regulatory roles for this enzyme in lipid metabolism.