Nitrate (NO3−) is known to be actively involved in the processes of mineralization and heavy metal transformation; however, it is unclear whether and how it affects the bioavailability of antimony (Sb) in paddy soils and subsequent Sb accumulation in rice. Here, the effects of NO3− on Sb transformation in soil-rice system were investigated with pot experiments over the entire growth period. Results demonstrated that NO3− reduced Sb accumulation in brown rice by 15.6% compared to that in the control. After amendment with NO3−, the Sb content in rice plants increased initially and then gradually decreased (in roots by 46.1%). During the first 15 days, the soil pH increased, the oxidation of Sb(III) and sulfides was promoted, but the reduction of iron oxide minerals was inhibited, resulting in the release of adsorbed and organic-bound Sb from soil. The microbial arsenite-oxidizing marker gene aoxB played an important role in Sb(III) oxidation. From days 15 to 45, after NO3− was partially consumed, the soil pH decreased, and the reductive dissolution of Fe(III)-bearing minerals was enhanced; consequently, iron oxide-bound Sb was transformed into adsorbed and dissolved Sb species. After day 45, NO3− was completely reduced, Sb(V) was evidently reduced to Sb(III), and green rust was generated gradually. Thus, the available Sb decreased due to its enhanced affinity for iron oxides. Moreover, NO3− inhibited the reductive dissolution of iron minerals, which ultimately caused low Sb availability. Therefore, NO3− can chemically and biologically reduce the Sb availability in paddy soils and alleviate Sb accumulation in rice. This study provides a potential strategy for decreasing Sb accumulation in rice in the Sb-contaminated sites.