Updated project metadata. In this study, we present a first proteomic overview of macadamia nut using a label-free shotgun proteomic approach and prediction of their proteins with potential allergenic activity and cross-reactivity via an in silico analysis. The defense response proteins were the most abundant group of proteins and accounted for 24% of the total abundance. The vicilin-like macadamia antimicrobial peptides 2-3 (MiAMP2) was the most abundant protein. The cascading of amino acid and carbohydrate metabolic pathways in macadamia nut were constructed and proposed for the first time. The in silico analysis revealed seed storage proteins (conglutins and vicilins), rubber elongation factor proteins, phosphate binding proteins, and detoxifying methylglyoxalases among others as proteins with allergenic and cross-reactive potential. The label-free shotgun proteomics study revealed valuable insight into the genetic and biological makeup of macadamia nut and potential proteins with predictive allergenic activity and cross-reactivity. Integration of genomics with the proteomic data will be indispensable for future molecular characterization of macadamia including allergens.