AbstractMicrobial dissimilatory sulfate reduction is a key process in the global sulfur and carbon cycles in anoxic ecosystems. In this anaerobic respiration, sulfate is phosphorylated and reduced to sulfite, which is further reduced to a DsrC‐trisulfide by the dissimilatory sulfite reductase DsrAB. DsrD is a small protein that acts as an allosteric activator of DsrAB, increasing the efficiency of sulfite reduction. Here, we report a detailed study of DsrD and its interaction with DsrAB. Sequence similarity analyses show that there are three groups of DsrD in organisms with a reductive‐type DsrAB. The protein regions involved in the DsrD–DsrAB interaction and activity‐promoting effect were investigated through in vitro and in silico studies, including mutations of conserved DsrD residues. The results reveal that the conserved β‐loop of DsrD is involved in the interaction, contributing to a better understanding of its mechanism of action.