[Objective] To investigate the effect of mediator subunit Med15B mediated fatty acid metabolism on Candida glabrata low pH tolerance. [Methods] Overexpression of genes elo1 and ole1, encoding fatty acid elongase and Δ9 desaturase, in strain Δmed15B we obtained an overexpressed strain Δmed15B/elo1-ole1. The expression level of genes elo1 and ole1, the membrane fatty acid composition, the membrane integrity and stress tolerance of parent strain ATCC55, strain Δmed15B and overexpression strain Δmed15B/elo1-ole1 were determined and compared at pH 2.0 and pH 6.0. [Results] At pH 2.0, the expression level of genes elo1 and ole1 in overexpression strain Δmed15B/elo1-ole1 was increased by 4.1-fold and 3.3-fold compared to those of strain Δmed15B, and increased by 2.5-fold and 2.2-fold compared to those of parent strain ATCC55, respectively. The average acyl-chain length of overexpression strain Δmed15B/elo1-ole1 reached 17.4 longer than that of the deletion strain (16.8) and parent strain (17.1), and the ratio of unsaturated fatty acid to saturated fatty acid (UFA/SFA ratio) increased by 53.1% and 41.5%, respectively. Furthermore, the percentage of propidium iodide (PI)-stained cell in overexpression strain Δmed15B/elo1-ole1 was decreased by 60.8% and 37.7%, respectively, compared to that of strain Δmed15B and parent strain ATCC55. Similarly, overexpression strain Δmed15B/elo1-ole1 exhibited more tolerance to low pH and the half-maximal inhibition (IC₅₀) values for pH stress was reached pH 2.3, which was pH 3.7 of strain Δmed15B and pH 3.1 of parent strain ATCC55. [Conclusion] These results indicated that overexpression genes elo1 and ole1 in strain Δmed15B can enhance cell tolerance to low pH by increasing the content of long chain and unsaturated fatty acids.