Lipase AK was modified with short alkyl chains to form a highly organic soluble enzyme and was used to catalyze the synthesis of biodiesel from soybean oil in organic media. The effects of several key factors including water content, temperature, and solvent were examined for the solubilized enzyme in comparison with several other commercially available lipases. Whereas native lipases showed no activity in the absence of water, the organic soluble lipase demonstrated reaction rates of up to 33 g-product/g-enzyme h. The biocatalyst remains soluble in the biodiesel product, and therefore, there is no need to be removed because it is expected to be burned along with the diesel in combustion engines. This provides a promising one-pot mix-and-use strategy for biodiesel production.