The aim of this study was to investigate the effect of dietary peppermint extract (PE) on innate immunity and inflammatory responses in the spleen of broiler chickens under chronic heat stress. In order to further study the mechanism of the activation of innate immunity and inflammation induced by chronic heat stress and the regulatory effect of peppermint extract, we examined the spleen's histological change, the mRNA expression of major pattern recognition receptors (PRRs) (TLR2, TLR4, NOD1, MDA5 and DAI) and transcription factors (NF-κB, AP-1 and IRF3) and downstream inflammatory cytokines (IFN-α, IFN-β, IL-1β, IL-6 and TNF-α) of innate immune signaling pathways associated with heat stress in the spleen of broiler chickens. The results indicated that chronic heat stress damaged the spleen tissue. In addition, chronic heat stress induced the activation of innate immunity and inflammatory responses by increasing the mRNA expression of TLR2, TLR4 and DAI, mRNA expression of transcriptional factors (NF-κB, AP-1 and IRF3) and the concentration of downstream inflammatory cytokines in the spleen of broiler chickens. Dietary peppermint extract alleviated the damage of spleen tissue caused by chronic heat stress. In addition, peppermint extract reduced the mRNA expression of DAI, mRNA expression of transcriptional factors NF-κB, AP-1 and IRF3, and the concentration of inflammatory cytokines in the spleen of broiler chickens under chronic heat stress. In conclusion, dietary peppermint extract could have a beneficial effect on regulating inflammatory response and innate immunity via inhibiting the activation of NF-κB, AP-1 and IRF3 signaling pathways mediated by DAI in the spleen of broiler chickens induced by chronic heat stress.