Candida antarctica lipase B (Cal-B) is one of the most recognized biocatalysts because of its high degree of selectivity in a broad range of synthetic applications of industrial importance. Herein, the substituent effects involved in transesterification catalyzed by Cal-B are explored in detail using a combination of experimental analysis and theoretical modeling. The transesterification ability of Cal-B was experimentally determined with 22 vinyl ester analogs and ribavirin as substrates and, on this basis, a series of quantitative structure-activity relationship (QSAR) models are developed using various structural parameters characterizing the variation in substituent groups of the substrate molecules. The resulting models exhibit a good stability and predictive power, from which five most important properties are highlighted and engaged to ascertain the structural basis and reaction mechanism underlying the transesterification. From the modeling analysis it is seen that the size, geometry, and charge distributions of substrate exert a significant effect on reaction yield, where, the size of the substituent group was the most significant impact factor on the reaction yield, the charge distribution was the second, and then the topological structure of the substrate.