Abstract Beauvericin (BEA), a cyclic depsipeptide, is a mycotoxin of the enniatin family and the secondary metabolite of various toxigenic fungi. Multiple biological functions of BEA have been well investigated, such as anti‐cancer, anti‐inflammatory, anti‐microbial, and immune‐activating functions. In a recent study, we showed that BEA can target Toll‐like receptor 4 (TLR4) to induce dendritic cell (DC) activation. In an in silico screen, we identified Cathepsin B (CTSB) as a potential additional interaction partner for BEA, which has been verified recently in a study showing inhibition of human CTSB activity by BEA in cell‐free assays. The underlying molecular mechanism of BEA‐mediated CTSB inhibition remains unknown, as do the cellular entities where this inhibition takes place. In this study, we determine the effects of BEA on CTSB within granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐cultured bone marrow‐derived dendritic cells (BMDCs) and human leukemia monocytic cell line THP‐1 induced immature dendritic cells (iDCs). BEA significantly suppresses CTSB activity in both mouse BMDCs and human iDCs. NMR analyses indicate that BEA directly interacts with CTSB. Enzyme kinetics show that BEA can directly inhibit CTSB activity and acts as an uncompetitive inhibitor. Molecular docking analysis revealed a putative binding site for BEA in human CTSB. Collectively, our study is the first to describe the molecular mechanisms underlying the biological activity of BEA against human CTSB, suggesting that CTSB may be a candidate target for tumor therapy.