Proteomics is a powerful approach to study the molecular mechanisms of cancer. In this study, we aim to characterize the proteomic profile of gastric cancer (GC) from patients with diabetes mellitus (DM). Forty gastric cancer tissue samples including 19 cases from diabetic patients, and 21 cases from individuals without diabetes (control group), were selected for proteomics analysis. Gastric tissues were lysed, sonicated and the resulting protein content was determined. One hundred g of proteins from each sample were processed following the single-pot, solid-phase-enhanced sample preparation approach – SP3 and enzymatically digested with trypsin. The resulting peptides were analyzed by LC-MS mass spectrometry. Comparison of protein expression levels between GC samples from diabetic and non-diabetic patients was performed by label-free quantification - LFQ. A total of 6599 protein groups were identified among the 40 samples. Two hundred-twenty proteins with at least two unique peptides were differentially expressed among the two groups (adjusted p-value < 0.05), 102 being upregulated and 118 downregulated in the diabetic cohort. Statistical overrepresentation tests were considered for different annotation sets including GO, KEGG, Reactome including Disease functional databases. Upregulated proteins in GC samples from diabetic patients showed a strong fold enrichment associated with respiratory electron transport and alcohol metabolic biological processes, while downregulated proteins were associated with cell-cell junction cellular components. Six proteins were exclusively detected in GC samples from diabetic patients, while 11 proteins were found to be absent in this group.