Microbially mediated Fe(II) oxidation is prevalent and thought to be central to many biogeochemical processes in paddy soils. However, we have limited insights into the Fe(II) oxidation process in paddy fields, considered the world's largest engineered wetland, where microoxic conditions are ubiquitous. In this study, microaerophilic Fe(II) oxidizing bacteria (FeOB) from paddy soil were enriched in gradient tubes with FeS, FeCO3, and Fe3(PO4)2 as iron sources to investigate their capacity for Fe(II) oxidation and carbon assimilation. Results showed that the highest rate of Fe(II) oxidation (k = 0.836 mM d−1) was obtained in the FeCO3 tubes, and cells grown in the Fe3(PO4)2 tubes yielded maximum assimilation amounts of 13C-NaHCO3 of 1.74% on Day 15. Amorphous Fe(III) oxides were found in all the cell bands with iron substrates as a result of microbial Fe(II) oxidation. Metagenomics analysis of the enriched microbes targeted genes encoding iron oxidase Cyc2, oxygen-reducing terminal oxidase, and ribulose-bisphosphate carboxylase, with results indicated that the potential Fe(II) oxidizers include nitrate-reducing FeOB (Dechloromonas and Thiobacillus), Curvibacter, and Magnetospirillum. By combining cultivation-dependent and metagenomic approaches, our results found a number of FeOB from paddy soil under microoxic conditions, which provide insight into the complex biogeochemical interactions of iron and carbon within paddy fields. The contribution of the FeOB to the element cycling in rice-growing regions deserves further investigation.