Toll-like receptor (TLR) signaling plays a key role in the intestinal innate and adaptive immune responses, and pregnancy has effects on intestinal length and villus height. Nevertheless, it is unclear if early pregnancy modulates the expression of TLR members in the duodenum of ewes. In this study, ovine duodenums were obtained at day 16 of the estrous cycle (N16), and days 13, 16, and 25 of gestation (P13, P16, and P25) from ewes, and mRNA and protein expression of TLR members were analyzed. The results showed that early pregnancy increased the expression of TLR3, TLR5, and TRAF6, but did not influence TLR4 expression. In addition, there were increases in expression of TLR2, MyD88, and IRAK1 at P13 and P16, or only at P16 compared to N16 and P25. In summary, these data suggest that early pregnancy modulates the TLR signal pathway in the maternal duodenum, which may be related to the immunoregulation and adaptation of the maternal duodenum in the ovine.