The sophorolipid-producing strain Starmerella bombicola CGMCC 1576 has a remarkable ability to produce sophorolipids (SLs) under the acidic and lactonic forms with almost equal proportion. In this study, we found the gene encoding for the long-chain acyl-CoA synthetase (ALCS). This enzyme was putatively identified as a membrane-bound long-chain fatty acid transport protein and contributed to the uptake of long-chain fatty acids. Disruption of the alcs gene resulted in an impaired growth of the alcs-deleted mutant in minimal media containing different fatty acids (C12:0, C14:0, C16:0, C18:0, C22:0, and C24:0) as the sole carbon source and led to a dramatic decrease in the uptake of the fluorescent-tagged long-chain fatty acid analogue-boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823). The absence of this alcs gene caused obvious phenotype changes. Compared with the wild-type strain, the yield and compositions of the SLs produced by the gene-deleted mutant of a dagger alcs::six showed almost no lactonic form of SLs, and the acidic SLs were composed of medium-chain. The ALCS enzyme was heterologously expressed in Escherichia coli JM109 (DE3) with pMAL-c2x-alcs. The enzyme was purified through a maltose-binding protein (MBP) affinity chromatography column and was confirmed to be homogeneous by SDS-PAGE. The recombinant enzyme could catalyze the formation of the long-chain acyl-CoA when the long-chain fatty acids and the coenzyme A were used as substrates.