The objective of the present study was to evaluate the effects of novel DNA vaccines fusing inhibin (INH) α (1-32) and RFamide-related peptide-3 (RFRP-3) genes on the immune response, reproductive hormone levels, and fertility of Tan sheep. Thirty-two female Tan sheep were divided into four groups (groups A, B, C, and D with 8 sheep per group) and respectively immunized (thrice, 20 d apart) with 0.6 mg of p-TPA-SINH/TPA-SRFRP (group A), p-SINH/SRFRP (group B), p-SINH (group C) or 0.4 ml saline (group D). Twenty days after primary immunization, all vaccines elicited significant immune responses, and the antibody levels of anti-INH and anti-RFRP-3 in the vaccinated groups were significantly higher (p < 0.05) than that of the control group. Immunization with p-TPA-SINH/TPA-SRFRP induced higher antibodies against INH and RFRP-3. Hormone levels of FSH and LH in group A immunized with p-TPA-SINH/TPA-SRFRP were significantly higher (p < 0.05) than those in group C, which are immunized with p-SINH and the control group 20 d after the third immunization. Additionally, the p-TPA-SINH/TPA-SRFRP, p-SINH/SRFRP, p-SINH and saline vaccine induced different twinning rate of ewes (37.5%, 37.5%, 12.5%, and 0, respectively), but no significant differences were found in improving twinning rate of ewes among four groups. These results suggested that neutralization of endogenous INH and RFRP-3 with novel DNA vaccine fusing INHα (1-32) and RFRP-3 genes successfully elicited a humoral immune response, increased reproductive hormone levels, but it did not significantly improve litter sizes and twinning rate of ewes in the present study. [ABSTRACT FROM AUTHOR]