Acyl-CoA synthetases (ACSs) are responsible for acyl-CoA synthesis from nonpolar hydrophilic fatty acids and play a vital role in many metabolic processes. As a category of ACS isozymes, members of ACS family (ACSF1-3) participate in lipid metabolism; however, their expression patterns, regulatory mechanisms and effects on egg-laying performance in chicken are poorly understood. Our in vivo and in vitro studies showed that ACSF1-3 genes were extensively expressed, and their expression levels changed dynamically in the liver among different development stages. Moreover, ACSF1 expression was upregulated and ACSF2 expression was downregulated by estrogen, but ACSF3 showed no response to estrogen treatment. The regulatory effect of estrogen on ACSF1 expression was mediated via ERα. The ACSF2 was highly expressed in the liver in peak-laying hens compared with pre-laying and late-laying hens, and also highly expressed in the liver continued egg-laying hens compared with inactive egg-laying hens. It is suggested that hepatic ACSF2 expression level might relate to egg-laying performance in chicken. In conclusion, the expression of ACSF1 was upregulated by estrogen via ERα, and the expression of ACSF2 was downregulated by estrogen and might be related to egg-laying performance in chicken.