Introduction Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. More than a decade ago it has been demonstrated that neuropeptide SIFamide targets the basal regions of saliva-producing type II and III acini via neuropeptidergic axons originating from giant protocerebral neurons in tick synganglion. Objectives •Molecular characterization and functional expression the Ixodes SIFamide receptors •Immuno-localization of SIFamide and its receptors in Ixodes salivary gland acini type II and III Material and methods •Molecular cloning •Phylogeny •Heterologous expression •Wholemount immunohistochemistry •Immunogold labelling transmission electron microscopy Results We identified two authentic Ixodes SIFamide receptors (SIFa_R1 and SIFa_R2) making this tick species the first arthropod in which two different SIFa_Rs have been functionally characterized. We also experimentally proved the high biological affinity of insect SIFamide paralogs to Ixodes SIFa_R2, but not SIFa_R1. The immunogold staining, in combination with transmission electron microscopy, revealed the expression of both SIFa_Rs on the basal epithelial cells and the single myoepithelial cell close to the apex of the acinar duct. As both these cell types overlay the arms of the acinar valve, we suggest that SIFamide and its two receptors regulate the release of saliva from the acinus to associated ducts by controlling this structure. In addition, the SIFa_R2, found to be expressed on the basal granular cells in both type II and III acini, likely control their secretory activities. Conclusion Our findings indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in Ixodes salivary glands. Here we highlight the structural and functional complexity of peptidergic control of Ixodes salivary glands, emphasizing its uniqueness among the arthropod phylum.