Hexavalent chromium [Cr(VI)] is a priority heavy metal pollutant causing a series of environmental issues, and bio-reduction of Cr(VI) to trivalent chromium can remarkably decrease the environmental risk of Cr(VI). The reduction and resistance abilities of microorganisms to Cr(VI) can be dramatically improved by acclimatization. In the present study, we collected Shewanella oneidensis MR-1 from a 120-day acclimatization by increasing Cr(VI) concentration in the culture media to investigate its adaptation mechanisms under long-term Cr(VI) stress at the proteome level. Tandem mass tag-based quantitative proteomic analysis was performed to study the differences between 9 collected samples. A total of 2500 proteins were quantified from 2723 identified protein groups. Bioinformatics analysis showed that the differentially expressed proteins after the 120-day Cr(VI) acclimatization were mostly related to flagellar assembly, ribosomes, transport, sulfur metabolism, and energy metabolism. The findings of this study present novel insights into the molecular mechanisms for the reduction and resistance of S. oneidensis MR-1 responding to long-term Cr(VI) stress at the proteome level.