Carboxypeptidase B (CPB) plays an important role in blood digestion in mosquitos, aiding the release of free amino acids. Anopheles CPB is a target to block malaria transmission because it facilitates Plasmodium invasion of the mosquito midgut. Our study aimed to discover inhibitors of Anopheles CPB to prevent Plasmodium development in the mosquito. The Anopheles gambiae cpb (Agcpb) gene without a signal sequence was cloned into the pET28b expression vector. The recombinant AgCPB protein was expressed in E. coli BL21(DE3) within inclusion bodies after induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The protein pellet was dissolved in 6 M urea, purified by affinity chromatography, and dialyzed in reaction buffer. The refolded recombinant AgCPB could digest the hippuryl-arginine substrate similarly to that of the commercial porcine pancreas CPB. The 20 top-scoring malaria box compounds from the virtual-screening results were then chosen for an in vitro inhibition assay against AgCPB. Four of the 20 malaria box compounds could inhibit AgCPB activity. The compound MMV007591 was the most potent inhibitor with an IC₅₀ at 0.066 µM. The results indicate that these candidate compounds may be utilized in drug development against mosquito CPB activity to curb malaria transmission.