Abstract A recombinant Pichia pastoris displaying Rhizomucor miehei Lipase (RML) on the cell surface was shown to be an efficient biocatalyst in biodiesel production from soybean oil. Various reaction parameters affecting the transesterification catalyzed by RML-displaying P. pastoris whole cells were studied. The optimal reaction conditions were: temperature 55°C, water activity 0.66, the ratio of isooctane to oil 2:1 (v/v) and the whole-cell biocatalyst 40% (w/w, soybean oil). To reduce the inactivation of displaying lipases by methanol, each 1.5 molar equivalent of methanol (mol/mol, soybean oil) were added in a three-step procedure at the reaction time of 0h, 24h and 48h, respectively. Under these optimized conditions, a methyl esters (ME) yield reached 83.14% after 72h, which significantly enhanced the ME yield catalyzed by the displaying lipase reported previously. The RML-displaying P. pastoris whole-cell biocatalyst exhibited quite a good operational stability with more than 80.46% of its original activity remained even after 10 batches reaction as well as storage stability. Thus, it showed promising potential in biodiesel production.