The southern pine beetle (Dendroctonus frontalis Zimmermann) kills all pines within its range and is among the most important forest pest species in the US. Using a specialized mycangium surrounded by gland cells in the pronotum, adult females culture, transport, and inoculate two fungi into beetle galleries during oviposition. These fungal symbionts, to varying degrees, exclude antagonistic fungi and provide nutrients to larvae. However, the mechanisms (e.g. secreted antibiotic chemicals or nutrients, proteins or pathways) by which this relationship is maintained are not known. Here we present the first global and differential proteome profile of the southern pine beetle pronotum. Two-dimensional polyacrylamide electrophoresis, tandem mass spectrometry, and database searches revealed that the majority of pronotal proteins were related to energy-yielding metabolism, contractile apparati, cell structure, and defence. The identified proteins provide important insights into the molecular and biochemical processes of, and candidates for functional genomics to understand mycangia and pronotum functions in, the southern pine beetle.