Abstract Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which, in turn, serves as the major acyl donor for various lipid metabolic pathways. Increasing the size of acyl-CoA pool by enhancing LACS activity appears to be a useful approach to improve the production and modify the composition of fatty acid-derived compounds, such as triacylglycerol. In the present study, we aimed to improve the enzyme activity of Arabidopsis thaliana LACS9 (AtLACS9) by introducing random mutations into its cDNA using error-prone PCR. Two AtLACS9 variants containing multiple amino acid residue substitutions were identified with enhanced enzyme activity. To explore the effect of each amino acid residue substitution, single-site mutants were generated and the amino acid substitutions C207F and D238E were found to be primarily responsible for the increased activity of the two variants. Furthermore, evolutionary analysis revealed that the beneficial amino acid site C207 is conserved among LACS9 from plant eudicots, whereas the other beneficial amino acid site D238 might be under positive selection. Together, our results provide valuable information for the production of LACS variants for applications in the metabolic engineering of lipid biosynthesis in oleaginous organisms.