Adequate ovarian hormones secretion is essential for pregnancy success. Oxidative damage and following inflammation can destroy the ovarian normal function in mammals. Daidzein (DAI) is a classical isoflavonic phytoestrogen with specific oestrogenic activity. This study aimed to explore the effects of daidzein supplementation on fertility and ovarian characteristics of sows through biochemical analysis and RNA-seq technology. Twelve multiparous Yorkshire × Landrace sows were randomly divided into CON and DAI groups. We found that DAI increased total number of embryos as well as P4 and E2 levels of serum. DAI not only elevated the activities of T-AOC and GSH-Px, but also tended to decrease the content of MDA and IL-6 in the serum. In ovary, RNA-Seq identified 237 differentially expressed genes (DEGs), and GO analysis showed that these DEGs were linked to functions associated with immune dysfunction. Moreover, STRING analysis demonstrated that most interacting nodes were TLR-4, LCP2, and CD86. Furthermore, DAI decreased the content of MDA, IL-1β, IL-6, and TNF-α, and increased the activities of T-AOC and CAT in ovarian tissue. Interestingly, a partial mantel correlation showed that T-AOC was the strongest correlation between the ovarian dataset and selected DEGs. Additionally, DAI supplementation not only increased the protein expressions of Nrf2, HO-1, and NQO1, but also decreased the protein expressions of TLR-4, p-NFκB, p-AKT, and p-IκBα. Altogether, our results indicated that DAI could ameliorate ovarian oxidative stress and inflammation in sows, which might be mediated by suppressing the TLR4/NF-κB signaling pathway and activating the Nrf2/HO-1 signaling pathway.