Whey, a by-product of cheese making, contains whey proteins, lactose, vitamins, and minerals. Whey and whey proteins are still not fully used. In this study, whey protein-based aqueous polymer-isocyanate (API) adhesives were developed and characterized by bond test, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscope (SEM) for bond strength, chemical structures, and morphology. The optimized whey protein-based API adhesive for Glulam had a 28-h boilingdry-boiling wet strength of 6.81 MPa and a dry strength of 14.34 MPa. Results indicated that the addition of polyvinyl acetate emulsion can prolong the work life of the API adhesive. Addition of crosslinker polymeric methylene bisphenyl diisocyanate (P-MDI) not only increased the cohesive strength of the cured adhesive by crosslinking whey proteins but also resulted in strong chemical bonds via urethane linkage in wood bondlines. Addition of polyvinyl alcohol (PVA) further increased the crosslinking density of the cured adhesive due to its capability of crosslinking whey proteins through the reaction with P-MDI. Nanoscale CaCO3 powder (3.5 wt %) as filler significantly improved bond strength due to its mechanical interlock with the polymers in the adhesive. SEM examinations confirmed that both PVA and nanoscale CaCO3 improved the compatibilities of the components in the optimized whey protein-based API adhesive. FTIR results revealed that P-MDI reacts mainly with the residual amino groups rather than the hydroxyl groups of whey proteins. (C) 2010 Wiley Periodicals, Inc. J Appl Polym Sci 120: 220-225, 2011