Transferrin (Tsf) and iron regulatory protein (IRP) are key iron metabolism-related moleculeswithwell-characterized functions in vertebrates, yet their roles and underlying mechanisms in ticks remain largely unexplored. This study provides the first comprehensive investigation of both Tsf and IRP in the hard tick Haemaphysalis longicornis, a major ectoparasite capable of transmitting various pathogens to humans and animals. Quantitative real-time PCR was employed to examine the dynamic expression profiles of Tsf and IRP genes across different developmental stages and various tissues of H. longicornis. Prokaryotic expression vectors pSMART-1- Tsf and pSMART-1- IRP were constructed, and expression conditions were optimized to obtain high- purity recombinant proteins through affinity chromatography. The functions of the recombinant proteins were subsequently characterized in vitro. RNA interference was performed to silence Tsf and IRP gene expression in H. longicornis to investigate the functions of these proteins in vivo. Furthermore, proteomic analysis of ovaries and midguts was conducted to elucidate the underlying molecular mechanisms. Expression analysis showed that both Tsf and IRP were transcriptionally active across all developmental stages (larva, nymph, adult female) and widely distributed in salivary glands, ovary, midgut, and Malpighian tubules, with significant upregulation after blood feeding. Recombinant Tsf and IRP exhibited iron chelation, radical- scavenging, and hemolytic activities in vitro; Tsf also displayed antibacterial activity, while IRP possessed aconitase enzymatic activity. RNA interference-mediated silencing of either gene significantly reduced engorgement weight, ovary weight, egg mass, and hatching rate, and prolonged egg incubation, demonstrating their essential roles in blood feeding and reproduction. Proteomic analysis revealed that Tsf and IRP may regulate tick physiology through convergent pathways, including ubiquitin-proteasome, lipid/carbohydrate metabolism, oxidative stress responses, mRNA surveillance, endocytosis, vesicle transport, and cell cycle regulation. These findings not only advance the fundamental understanding of iron metabolism in ticks but also suggest that both Tsf and IRP could serve as candidate antigens for developing anti-tick vaccines through the disruption of iron metabolic pathways.