Updated project metadata. Drug resistance in bacteria is becoming a significant threat to global public health, and the development of novel and efficient antibacterial compounds is urgently needed. Recently, rhodium complexes have attracted attention as antimicrobial agents, yet their antibacterial mechanism remains unknown. In this study, we observed that the dirhodium (II) complex Rh2Ac4 inhibited Streptococcus. pneumoniae growth without significant cytotoxic side-effects on host cells in vitro. We subsequently investigated the antibacterial mechanism of Rh2Ac4 using iTRAQ-based proteomics combined with cellular and biochemical assays. Bioinformatics analysis on the proteomic alterations demonstrated that six molecular functional groups, including metal ion binding and twelve metabolic pathways, were significantly affected after treatment with Rh2Ac4. The interaction network analysis of metal ion binding proteins suggested that Rh2Ac4 decreased the protein expression levels of SPD_1652, SPD_1590 and Gap, which are associated with haem uptake/metabolism. Cellular and biochemical assays further confirmed that Rh2Ac4 could be taken up by bacteria via the PiuABCD haem-uptake system. The structurally similar Rh complex may compete with Fe-haem to decrease Fe-uptake via the PiuABCD system, disrupting iron metabolism to exert its antibacterial activity against S. pneumoniae. These data indicate that Rh2Ac4 is a promising new drug for the treatment of S. pneumoniae infections.