Crustacean hyperglycemic hormone (CHH) has gained wide attention for its diverse physiological functions, including in metabolism and reproduction. In this study, cDNA sequence encoding CHH was cloned from the brain and ventral nerve cord (VNC) of the mud crab, Scylla olivacea. CHH of S. olivacea (Scyol-CHH) consists of 642 nucleotides, which encode a 108-amino acid CHH prepropeptide. Based on in situ hybridization, Scyol-CHH was expressed in the neurons within the eyestalk, including at neuronal clusters 1–4. Scyol-CHH was also detected in the neurons within the neuronal clusters 6 and 8–11 of the brain. In the VNC, its expression was found in the neurons within the subesophageal ganglion as well as the abdominal ganglion. We found that administration of serotonin (5-HT) significantly increased the expression of Scyol-CHH in the brain and VNC at 3 and 6 h post-injections, respectively, as compared with the controls. This suggests that 5-HT may play an important role in the regulation of CHH expression in the nervous system. Moreover, injection of 5-HT up-regulated Scyol-CHH expression in ovary, suggesting a reproductive-related role for 5-HT and CHH in S.