Neuromedin U (NMU) and its structurally-related peptide, neuromedin S (NMS), are reported to regulate many physiological processes and their actions are mediated by two NMU receptors (NMUR1, NMUR2) in mammals. However, the information regarding NMU, NMS, and their receptors is limited in birds. In this study, we examined the structure, functionality, and expression of NMS, NMU, NMUR1 and NMUR2 in chickens. The results showed that: 1) chicken (c-) NMU cDNA encodes a 181-amino acid precursor, which may generate two forms of NMU peptide with 9 (cNMU-9) and 25 amino acids (cNMU-25), respectively. 2) Interestingly, two cNMS transcripts encoding two cNMS precursors of different lengths were identified from chicken pituitary, and both cNMS precursors may produce a mature cNMS peptide of 9 amino acids (cNMS-9). 3) cNMU-9, cNMU-25 and cNMS-9 could activate cNMUR1 expressed in HEK293 cells potently, as monitored by three cell-based luciferase reporter systems, indicating that cNMUR1 can act as a receptor common for cNMU and cNMS peptides, whereas cNMUR2 could be potently activated by cNMS-9, but not by cNMU-9/cNMU-25. 4) cNMU and cNMUR1 are widely expressed in chicken tissues with abundant expression noted in the gastrointestinal tract, as detected by quantitative real-time PCR, whereas cNMUR2 expression is mainly restricted to the brain and anterior pituitary, and cNMS is widely expressed in chicken tissues. Collectively, our data helps to elucidate the physiological roles of NMU/NMS peptides in birds and reveal the functional conservation and changes of NMU/NMS-NMUR axis across vertebrates.