Mammalian carboxylesterases (CES) are key enzymes that participate in the hydrolytic metabolism of various endogenous and exogenous substrates. Human carboxylesterase 2A (hCES2A), mainly distributed in the small intestine and colon, plays a significant role in the hydrolysis of many drugs. In this study, 3-arylisoquinolones 3 h [3-(4-(benzyloxy)-3-methoxyphenyl)-7,8-dimethoxyisoquinolin-1(2H)-one] and 4 a [3-(4-(benzyloxy)-3-methoxyphenyl)-4-bromo-7,8-dimethoxyisoquinolin-1(2H)-one] were found to have potent inhibitory effects on hCES2A (IC₅₀=0.68 μΜ, K_i=0.36 μΜ) and excellent specificity (more than 147.05-fold over hCES1 A). Moreover, 4 a exhibited threefold improved inhibition on intracellular hCES2A in living HepG2 cells relative to 3 h , with an IC₅₀ value of 0.41 μΜ. Results of inhibition kinetics studies and molecular docking simulations demonstrate that both 3 h and 4 a can bind to multiple sites on hCES2A, functioning as mixed inhibitors. Structure−activity relationship analysis revealed that the lactam moiety on the B ring is crucial for specificity towards hCES2A, while a benzyloxy group is optimal for hCES2A inhibitory potency; the introduction of a bromine atom may enhance cell permeability, thereby increasing the intracellular hCES2A inhibitory activity.