It is well documented that vasoactive intestinal peptide (VIP) is an avian prolactin-releasing factor, regulating the reproductive cycle, that initiates and maintains incubation behavior in avian species. Native Thai chicken has strong maternal behaviors. Thus, it is an excellent animal model to study this phenomenon. Changes in the numbers of VIP neurons within the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) are associated with maternal behaviors in the native Thai chicken. The objective of this study was to elucidate the role of VIP during the transition from egg incubation to rearing behavior in this species. The distributions of VIP-immunoreactive (-ir) neurons and fibers within the nucleus septalis lateralis (SL), nucleus anterior medialis hypothalami (AM), regio lateralis hypothalami (LHy), nucleus commissurae pallii (nCPa), nucleus ventromedialis hypothalami (VMN), and IH-IN were compared between incubating (INC) hens and replaced-egg-with-chicks (REC) hens, in which eggs were replaced by 3-day-old chicks after 6, 10, and 14 d of incubation period for 3 days. Using an immunohistochemistry technique, the results revealed that VIP-ir neurons and fibers were found within the SL, AM, LHy, nCPa, VMN, and IH-IN, with the greatest expression observed in the IH-IN in both groups. The number of VIP-ir neurons in the IH-IN was markedly decreased (P < 0.05) in the REC hens when compared with those of the INC hens at 3 different time points (at d 9, 13, and 17). However, changes in the number of VIP-ir neurons between the INC and REC hens were not observed within other hypothalamic areas tested. These findings indicate that the presence of eggs and chicks affects the VIPergic system, and VIP neurons in the IH-IN might play a role in the transition from incubating to rearing behavior in the native Thai chicken.