In this study, lyophilized Pseudomonas aeruginosa based whole-cell biocatalyst was used in the highly efficient and regioselective acylation of helicid with fatty acid vinyl esters in organic solvents. P. aeruginosa whole-cell catalyst showed high biomass and biocatalytic performance using 0.1% (w/v) soybean oil and 0.2% (w/v) tryptone as the carbon and nitrogen source, respectively. The biomass (0.72 g/L) cultivated for 36 h under the optimized conditions was 2-folds higher than that obtained by the use of the initial conditions (0.36 g/L). In addition, the optimized medium was found to be suitable for P. aeruginosa cultured in 3.5-L scale fermentation with high biomass (0.71 g/L) obtained at 24 h. Besides, a high substrate conversion efficiency (ranged from 92 to 99%) and excellent regioselectivity (99%) was achieved when P. aeruginosa whole-cell biocatalyst was applied to synthesize an array of 6 '-esters of helicid. The reaction rate varies with varying acyl donors because of their differential interactions with the catalytic site of the enzyme from the whole cells of P. aeruginosa. Taken together, P. aeruginosa whole-cell catalyst obtained using optimal culture medium manifest a promising and highly-regioselective alternative to purified enzymes for the regioselective formation of 6 '-esters derivatives of helicid in non-aqueous solvents. [GRAPHICS] .