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Halomonas spp. are moderately halophilic bacteria with the ability to tolerate various heavy metals. However, the role of basic cellular metabolism, particularly amino acid metabolism, has not been investigated in Halomonas spp. under excess Mn(Ⅱ). The strain Halomonas sp. MNB13 was isolated from a deep-sea ferromanganese nodule and can tolerate 80 mM Mn(Ⅱ). To comprehensively explore the mechanisms underlying its resistance to excess Mn(Ⅱ), we conducted a comparative proteome analysis. The data revealed that both 10 mM and 50 mM Mn(Ⅱ) significantly up-regulated the expression of proteins involved in Mn(Ⅱ) transport (MntE), oxidative stress response (alkyl hydroperoxide reductase and the Suf system), and amino acid metabolism (arginine, cysteine, methionine, and phenylalanine). We further investigated the role of cysteine metabolism in Mn(Ⅱ) resistance by examining the function of its downstream product, H 2 S. Consistent with the up-regulation of cysteine desulfurase, we detected an elevated level of H 2 S in Halomonas sp. MNB13 cells under Mn(Ⅱ) stress, along with increased intracellular levels of H 2 O 2 and O 2 •−. Upon exogenous addition of H 2 S, we observed a significant restoration of the growth of Halomonas sp. MNB13. Moreover, we identified decreased intracellular levels of H 2 O 2 and O 2 •− in MNB13 cells, which coincided with a decreased formation of Mn-oxides during cultivation. In contrast, in cultures containing NaHS, the residual Mn(Ⅱ) levels were higher than in cultures without NaHS. Therefore, H 2 S improves Mn(Ⅱ) tolerance by eliminating intracellular reactive oxygen species rather than decreasing Mn(Ⅱ) concentration in solution. Our findings indicate that cysteine metabolism, particularly the intermediate H 2 S, plays a pivotal role in Mn(Ⅱ) resistance by mitigating the damage caused by reactive oxygen species. These findings provide new insights into the amino acid mechanisms associated with Mn(Ⅱ) resistance in bacteria. • High levels of Mn(Ⅱ) affected the metabolism of certain amino acids in MNB13. • Cysteine desulfurase responsible for H 2 S production was significantly up-regulated. • Increased amounts of H 2 S, H 2 O 2 , and O 2.•− were detected in MNB13 under Mn(Ⅱ) stress • H 2 S restored the growth of MNB13 and reduced H 2 O 2 and O 2.•− intracellular levels • H 2 S decreased the formation of Mn(Ⅱ)-oxides by MNB13. [ABSTRACT FROM AUTHOR]