Apolipophorin III (apoLp-III) is a multifunctional protein that is involved in lipid transport and innate immunity in insects. In this study, an apoLp-III protein that exhibits antibacterial activity was identified in honeybees (Apis cerana). A. cerana apoLp-III cDNA consists of 193 amino acids that share high protein sequence identity to other members of the hymenoptran insect apoLp-III family. A. cerana apoLp-III is expressed constitutively in the fat body, epidermis, and venom gland and is detected as a 23-kDa protein. A. cerana apoLp-III expression is induced in the fat body after injection with Escherichia coli, Bacillus thuringiensis, or Beauveria bassiana. However, recombinant A. cerana apoLp-III (expressed in baculovirus-infected insect cells) binds directly to E. coli and B. thuringiensis but not to B. bassiana. Consistent with these findings, A. cerana apoLp-III exhibited antibacterial activity against Gram-negative and Gram-positive bacteria. These results provide insight into the role of A. cerana apoLp-III during the innate immune response following bacterial infection.