A prodrug is a pharmacologically inactive derivative of an active parent drug, and is bioconverted to the active drug in vivo. Through chemical modification of a drug to a prodrug, we are able to deliver drugs to the target site, to optimize therapy and minimize toxicity. A major pathway for the bioconversion of prodrugs to the active parent drugs is via carboxylesterase (CES) activity. Among human CES isozymes, hCE1 and hCE2 predominantly participate in the hydrolysis of prodrugs in the liver and small intestine, respectively, although the substrate specificity is quite different between two isozymes; therefore, we can rationally design prodrugs based on the enzyme characteristics. However, since the expression levels of CES vary among individuals, there is a range of pharmacological responses following prodrug administration. Species differences are caused by tissue-dependent hydrolase activity mediated by CES, which makes it difficult to predict effectiveness in humans from a preclinical study using animals. Accordingly, understanding the regulation of CES expression and species difference of CES catalytic properties will be helpful in the design of prodrugs with increased specificity and enhanced physicochemical and biological properties.