To examine the feasibility of chitosan as an alternative transfection reagent candidate for protein expression in Bm5 cells and silkworm larvae using recombinant BmNPV bacmid DNA. Chitosan 100 and recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA, in amino group/phosphate group (N/P) ratios of 0.1-10, were used for formation of chitosan/DNA nanocomplexes. The chitosan/BmNPV bacmid DNA nanocomplexes showed higher specific activity of GFP(uv)-beta 1,3-N-acetylglucosaminyltransferase 2 (beta 3GnT2) fusion protein (GGT2) expressed in silkworm larvae than DMRIE-C, a conventional silkworm transfection reagent. In particular, the composition of chitosan and BmNPV bacmid DNA nanocomplexes formed by an N/P ratio of 8 or 10, respectively, showed the highest specific activity of beta 3GnT2 in the silkworm larvae hemolymph. In addition, three different proteins were expressed in silkworm larvae to the same extent using chitosan as that using DMRIE-C. This is the first finding that chitosan/BmNPV bacmid DNA nanocomplexes can rival the performance of commercially available transfection reagents for the expression of recombinant proteins in Bm5 cells and silkworm larvae.