Assimilatory nitrite reductase (aNiR) reduces nitrite ions (NO$\rm{{_{2}^{-}}}$) to ammonium ions (NH$\rm{{_{4}^{+}}}$), whereas assimilatory sulfite reductase reduces sulfite (SO$\rm{{_{3}^{2-}}}$) to hydrogen sulfide (HS−). Although aNiR can also reduce SO$\rm{{_{3}^{2-}}}$, its activity is much lower than when NO$\rm{{_{2}^{-}}}$ is reduced as the substrate. To increase the SO$\rm{{_{3}^{2-}}}$‐reduction activity of aNiR, we performed a N226K mutation of Nii3, a representative aNiR. The resulting Nii3‐N226K variant could bind non‐native targets, SO$\rm{{_{3}^{2-}}}$, and HCO$\rm{{_{3}^{-}}}$, in addition to its native target, i.e., NO$\rm{{_{2}^{-}}}$. We have determined the high‐resolution structure of Nii3‐N226K in its apo‐state and in complex with SO$\rm{{_{3}^{2-}}}$, NO$\rm{{_{2}^{-}}}$, and HCO$\rm{{_{3}^{-}}}$. This analysis revealed conformational changes of Lys226 and the adjacent Lys224 upon binding of SO$\rm{{_{3}^{2-}}}$, but not NO$\rm{{_{2}^{-}}}$. In contrast, HCO$\rm{{_{3}^{-}}}$ binding induced a conformational change at Arg179. After replacing Asn226 with a positively charged Lys, aNiR showed affinity for several anions. A comparison of all ligand‐bound structures for Nii3‐N226K revealed that structural changes in the active site depend on the size of the substrate.