Abstract: This study was conducted to investigate the regulatory mechanisms of egg laying and development of incubation behaviour by immunization against vasoactive intestinal peptide (VIP) in hens. In experiment 1, 18wk old prelay Taihe pullets were immunized on d1, d28, d56 and d84 of the experiment, with VIPBSA conjugate immunogen. In experiment 2, laying 23wk old Yuehuang hens were immunized on d1, d23, d48, d73 and d98 of the experiment, with tethered 4VIP recombinant protein as immunogen. Tissues of hypothalamus, pituitary gland, theca and granulosa layers of F1 to F3 ovarian follicles were collected, in 10 immunized and 10 control hens after slaughter on d98. The results of experiment 1 showed that immunization against VIP significantly attenuat the prepeak egg laying during d55 to d61 of the experiment 1, but significantly slowed postpeak decline of egg laying from d89 to d103 of the experiment. Meanwhile immunization against VIP also delayed the development of incubation behaviour. In experiment 2, the initial egg laying rate within 30 days after the experiment was low in both VIPimmunized and control hens, but was significantly higher in the former than that in the latter. Following increase of photoperiod from 12 h to 16 h, laying rate increased to the peak in both groups of hens. However VIPimmunized hens exhibited a substantial decrease in egg laying, which was significantly lower than that in the control group from d80 to d105 of the experiment. Percentage of hens incubating increased at a slower rate in the VIPimmunized hens than that in the control group, but were similar between the two groups at the end of the experiment. On d98 of the experiment, both groups of hens had similar numbers of large yellow, small yellow and large white follicles on the ovary. Immunization against VIP did not affect the mRNA expression level of GnRHⅠ, VIP in the hyothalamus, nor affected LHβ and FSHβ in the pituitary gland. However, PRLRmRNA expression level in both hypothalamus and pituitary, and PRL expression level in the pituitary gland were all significantly lower in VIPimmunized hens. In addition, immunization against VIP decreased PRLR mRNA expression in granulosa cells of F1 and F3 follicles, and decreased LHR mRNA level in granulose and theca layers of F1F3 follicles examined. Results of the 2 experiments demonstrated that immunization against VIP decreased gene expression and therefore secretion of PRL in the pituitary gland, which subsequently slowed development of incubation occuring after egg laying. Suppression of PRL secretion following VIPimmunization also depresses egg laying at certain stages, which may be a result of decreased LHR mRNA expression in the ovarian follicular cells.