Heavy metal accumulation in agricultural areas is a global environmental problem that affects microorganisms and plants, with serious implications for human health. This study aimed to investigate the molecular responses of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5 to cobalt stress. We evaluated bacterial growth and cell viability under cobalt stress and performed comparative proteomic and reverse genetics analyses. Cobalt significantly inhibited bacterial growth but did not cause cell death. Proteomic analysis in the presence of 2.5 mM CoCl2, which caused approximately 50% growth inhibition, revealed the induction of pathways related to iron uptake, carbohydrate metabolism, amino acid metabolism, quality control, and efflux. Knockout mutants for genes involved in these pathways (∆tbdR, ∆zwf, ∆pdhB, ∆argH and ∆czcC) confirmed the essential role of the CzcC efflux system in cobalt tolerance. Cobalt stress triggers molecular responses in G. diazotrophicus PAL5, with efflux systems playing a crucial role in stress tolerance.