Osteomyelitis is the leading cause of non-traumatic lower limb amputation in patients living with diabetes. The pathophysiology of diabetic foot osteomyelitis (DFO) is complex and incompletely understood. Using FFPE bone biopsies obtained from patients with suspicious clinical presentation of DFO, we characterized the proteome of bone infection in the diabetic microenvironment. A discovery LC/MS approach was used to identify molecules of interest in subjects diagnosed with DFO versus no DFO based on histopathology findings. In total, 2,135 proteins were identified across 18 DFO and 12 non-DFO TMT labeled samples. After applying data filtering and imputing missing values, 668 proteins were retained for statistical analysis. Of 66 significant proteins (p<0.05) , 8 demonstrated twofold abundance decreases in DFO with respect to non-DFO samples, while 8 proteins were enriched by at least onefold. Ontological analysis suggested that proteins regulating chemokine signaling and integrin mediated cascades are reduced in DFO, potentially resulting in suppression of downstream MAPK pathways, and impairing inflammatory response and cellular adhesion. Our analysis demonstrates that archived FFPE samples are a rich source for elucidating molecular mechanisms associated with diabetic foot infection and may improve accuracy of diagnosis, contribute to novel therapeutic approaches, and uncover new disease biomarkers. Disclosure A.Sherwood: None. K.L.Rubitschung: None. P.Crisologo: None. H.Hwang: None. L.A.Lavery: Advisory Panel; Acera Surgical Inc., Uluru, Research Support; Integra, Smith+Nephew, Tissue tech. O.K.Oz: None. Funding American Diabetes Association (1-17-ICTS-056) ;