One attractive feature of the baculovirus-insect cell system (BICS) is the baculoviral genome has a large capacity for genetic cargo. This enables construction of viral vectors designed to accept multigene insertions, which has facilitated efforts to produce recombinant multisubunit protein complexes. However, the large genetic capacity of baculovirus vectors has not yet been exploited for multistep pathway engineering. Therefore, we created PolyBac, which is a novel baculovirus shuttle vector, or bacmid, that can be used for this purpose. PolyBac was designed to accept multiple transgene insertions by three different mechanisms at three different sites within the baculovirus genome. After constructing and characterizing PolyBac, we used it to isolate nine derivatives encoding various combinations of up to eight different protein N-glycosylation pathway functions, or glycogenes. We then used these derivatives, which were designed to progressively extend the endogenous insect cell pathway, to assess PolyBac's utility for protein glycosylation pathway engineering. This assessment was enabled by engineering each derivative to produce a recombinant influenza hemagglutinin (rH5), which was used to probe the impact of each glycoengineered PolyBac derivative on the endogenous insect cell pathway. Genetic analyses of these derivatives confirmed PolyBac can accept large DNA insertions. Biochemical analyses of the rH5 products showed each had distinct N-glycosylation profiles. Finally, the major N-glycan on each rH5 product was the predicted end product of the engineered N-glycosylation pathways encoded by each PolyBac derivative. These results generally indicate that PolyBac has utility for multistep metabolic pathway engineering and directly demonstrate that this new bacmid can be used for customized protein glycosylation pathway engineering in the BICS.