To develop a structural homolog of mannosylerythritol lipids (MELs), Pseudozyma tsukubaensis JCM16987 (known to be a specific producer of the diastereomer type of mono-acetylated MEL (MEL-B)) was cultivated in medium containing 4 % (w/v) olive oil as the primary carbon source and 4 % L-arabitol as the supplemental sugar alcohol. Based on thin-layer chromatography (TLC), the glycolipid extract showed two major spots corresponding to MEL-B and an unknown glycolipid (GL1). Based on high-performance liquid chromatography after acid hydrolysis, GL1 from the L-arabitol culture showed two primary peaks identical to mannose and arabitol using the sugar analysis column, and one peak identical to L-arabitol was detected using the chiral resolution column. Based on NMR analysis, GL1 was identified as mono-acetylated mannosyl-L-arabitol lipid (MLAL-B) consisting of mannose, with L-arabitol as the sugar moiety. The observed critical micelle concentration (CMC) and surface tension at the CMC (γCMC) of MLAL-B were 1.2 × 10(-5) M and 32.8 mN/m, which were significantly higher than MEL-B (CMC = 3.1 × 10(-6) M and γcmc = 26.1 mN/m). Furthermore, based on a water-penetration scan, MLAL-B efficiently formed lamellar phase (Lα) and myelins at a broad concentration range. Thus, the present glycolipid showed higher hydrophilicity and/or water solubility and increased our understanding of environmentally advanced biosurfactants.