As a centre enzyme in fatty acid activation, acyl-CoA synthetase long-chain family member 1 (ACSL1) plays an important role in body lipid homeostasis. However, the functions of in the subcutaneous adipogenesis of pigs are largely unknown. In the present study, we found that the expression of significantly increased during the process of porcine preadipocyte differentiation. Moreover, silencing of in preadipocytes decreased levels of triglyceride and adipogenic-related markers, including , , and ( < 0.01), and simultaneously increased levels of lipolytic-related markers, such as and ( < 0.05). Conversely, overexpression of in preadipocytes increased levels of triglyceride and , , and ( < 0.01), and reduced levels of and ( < 0.05). Luciferase reporter assays revealed that is a target of miR-218-5p, which can reduce the mRNA and protein levels of by directly binding the 3' untranslated region of . Furthermore, miR-218-5p has an inhibition role in porcine preadipocyte differentiation by suppressing expression. Taken together, these data provide insights into the mechanism of the miR-218-5p/ axis in regulating subcutaneous fat deposition of pigs.