Mammalian carboxylesterases play an important role in the hydrolysis of both endogenous substrates and xenobiotics bearing ester or amide bond(s). We previously reported that bysspectin A and its derivative LC-20W were potent reversible hCES2A inhibitors. Here, a series of bysspectin A derivatives were designed and synthesized using LC-20W as the leading compound. Compound 9d was identified as a potent serine-targeting covalent inhibitor of hCES2A (IC(50) = 0.12 nM), which was much more potent than that of LC-20W. Further chemoproteomics and docking simulations showed that 9d could selectively modify hCES2A at the catalytic serine (Ser228), thereby blocking its catalytic activity. Notably, 9d showed good cell-membrane permeability and was capable of inhibiting intracellular hCES2A in living cells. In vivo tests showed that 9d significantly alleviates irinotecan-induced diarrhea and dextran sulfate sodium-induced colitis. Collectively, a novel serine-targeting covalent inhibitor against hCES2A was developed, offering a promising candidate for treating drug-induced diarrhea and ulcerative colitis.