An iTRAQ-based comparative and quantitative proteomics analysis of a soil-borne Mn(II)-oxidizing bacterium, Escherichia coli MB266, was conducted during the exponential and stationary growth phases. A total of 1850 proteins were identified in 4 samples, of which 373 and 456 proteins were significantly up- or down-regulated in at least one pairwise comparison, respectively. The iTRAQ data indicated that several enzymes involved in fatty acid metabolism (i.e., FabA, FabD and FabZ) and pyruvate metabolism (particularly pyruvate oxidase PoxB) were significantly up-regulated, while those related to the tricarboxylic acid cycle (such as FrdB, FumB and AcnA) and methylcitrate cycle (i.e., PrpC) were inactivated in the presence of 1 mM Mn(II); the amounts of some stress response and signal transduction system-related proteins (i.e., Spy) were remarkably increased, and the cold shock protein CspD was significantly up-regulated during the exponential growth phase. However, all verified heat shock proteins remained unchanged. The reactive oxygen species response and some redox enzymes might also be involved in Mn oxidation processes. The involvement of several cellular proteins in Mn(II) oxidation, including PoxB, Spy and MCO266, was further confirmed by gene disruption and expression complementation experiments. Based on these results, a signal transduction mechanism coupled to Mn oxidation was proposed.