Updated project metadata. Background: Bee venom (Apis mellifera) is a complex biological secretion rich in peptides, enzymes, and bioactive molecules with recognized pharmacological potential. Environmental factors may influence venom composition, yet geographic variation in the bee venom proteome remains insufficiently characterized, particularly in Mediterranean ecosystems of South America, including Chile. Methods: Venom samples obtained from multiple colonies of Apis mellifera within each geographic area in the Valparaíso Region of central Chile, were pooled to generate one composite biological sample per zone: Sea Level (SL), Inland Valley (IV), and Andean Foothills (AF). Two analytical aliquots from each pooled sample were processed independently for analysis. Proteomic profiling was performed using chromatography–tandem mass spectrometry (LC–MS/MS) with a data-dependent acquisition workflow. Label-free quantification based on spectral counts was used to assess differential protein expression across zones. Results: A total of 679 proteins were identified across all LC–MS/MS runs, including 604 proteins detected in SL samples, 530 in IV, and 395 in AF samples, with 360 proteins consistently quantified across the analytical duplicates from the three geographic zones and included in pairwise comparisons. Approximately 25–28% of quantified proteins were differentially expressed (DEPs) across pairwise analytical comparisons, suggesting geographically associated variation in venom composition. SL samples exhibited relative enrichment of classical venom components, including melittin, phospholipase A2, and venom-associated enzymes, consistent with a more toxin-oriented proteomic profile. In contrast, AF and IV samples showed increased abundance of proteins associated with stress response, metabolism, and secretion, including heat shock proteins, redox enzymes, and major royal jelly proteins (MRJPs), suggesting a shift toward physiological adaptation and glandular secretion-related processes. Conclusion: These findings suggest that bee venom proteome may vary across environmental gradients, reflecting both conserved and context-dependent components. The observed shift between toxin-dominated and stress-associated proteomic profiles is consistent with the concept of environmentally associated plasticity in venom composition. This variability has important implications for the pharmacological use and standardization of bee venom, highlighting the need to consider geographic origin in biomedical applications. Further studies integrating ecological, biochemical, and pharmacological approaches are needed to evaluate the functional and translational relevance of these proteomic differences.