Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts. Mmf1, a putative transporter of MELs, is conserved in the MEL biosynthesis gene clusters of diverse MEL producers, including the genera Ustilago, Pseudozyma, Moesziomyces, and Sporisorium. To clarify the function of Mmf1, we generated the gene-deleted strain of P. tsukubaensis Delta PtMMF1 and evaluated its MEL production. Using thin-layer chromatography analyses, we detected most MELs produced by Delta PtMMF1 in the culture supernatant. The spot size of diacylated MEL-B (the only product of the parental strain) was significantly smaller for strain Delta PtMMF1 than for the parental strain, and a mono-acylated MEL-D spot was detected. In addition, an unknown glycolipid was detected in the sample extracted from strain Delta PtMMF1. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that the unknown glycolipid was a novel MEL homologue, mono-acylated MEL-B.