Starmerella bombicola is a non-conventional yeast commercially used as a microbial cell factory for sophorolipid production. Sophorolipids are glycolipid biosurfactants composed of a glucose disaccharide sophorose and a fatty acid. In de novo sophorolipid synthesis, the fatty acid moiety is derived from the fatty acid synthesis (FAS) complex; therefore, the yeast's lipid metabolism plays a crucial role in sophorolipid biosynthesis. As a fatty acid precursor, citric acid is a key primary metabolite that connects carbohydrate and lipid metabolism, and in S. bombicola, it also has a regulatory effect on sophorolipid composition and productivity. We aimed to identify the mitochondrial transporters involved in citrate shuttling and the ATP citrate lyase (Acl), the enzyme that converts citric acid into acetyl-CoA. Subsequently, we studied their role in the citric acid shuttle and glycolipid synthesis and the potential of citrate metabolism as a genetic manipulation target for increased glycolipid synthesis. Bioinformatics analyses predicted 32 mitochondrial carriers of which two were identified as citrate transporters, named SbCtp1 and SbYhm2. Deletion of these mitochondrial carriers led to a lesser sophorolipid yield and a shift in the lactonic/acidic sophorolipid ratio. However, only the knockout of SbYhm2 caused a decrease of citric and an increase of malic acid extracellular concentrations. Additionally, deletion of SbAcl1 had a negative effect on S. bombicola's specific growth rate and sophorolipid synthesis and contributed to extra- and intracellular citric acid accumulation. Unexpectedly, SbAcl1 overexpression also decreased glycolipid production.Key Points• Starmerella bombicola is an industrially relevant microbial cell factory for biosurfactant production.• There are 32 predicted mitochondrial carriers in S. bombicola.• Citrate mitochondrial carriers SbYhm2 and SbCtp1 are essential for glycolipid synthesis in S. bombicola.• Deletion of SbAcl1 negatively affects growth and sophorolipid production in S. bombicola. Graphical abstract.