Improving immune function is an important indicator for establishing cold adaptation in broilers. In the study, to explore the effects and molecular mechanisms of intermittent and mild cold stimulation (IMCS) on the immune function of broilers, CIRP and TRPM8, induced by cold stimulation, as well as the NF-κB and MAPK pathways which play an important role in immune response, were selected to investigate. A total of 192 one-day-old broilers (Ross 308) were selected and randomly divided into the control group (CC) and the cold stimulation group (CS). The broilers in CC were raised at normal feeding temperature from d 1 to 43, while the broilers in CS were subjected to cold stimulation from day 15 to 35, with a temperature 3 °C below that of the CC group for 5 h, at 1 d intervals. The results showed that IMCS had little effect on the broiler hearts, and the myocardial structure was not damaged. On d 22, IMCS significantly increased the mRNA levels of CIRP, TRPM8, P65, P38, COX-2, TNF-α, IFN- γ, IL-6, IL-10, and the protein levels of CIRP, P65, P38, IL-1β and iNOS in the hearts, and the levels of CIRP and all cytokines in the serum (P ≤ 0.05). The mRNA and protein levels of IκB-α were significantly reduced (P ≤ 0.05). On d 36, the mRNA levels of TRPM8, P65, ERK, and IL-10 in the hearts and the content of COX-2 in the serum in CS were increased significantly (P ≤ 0.05), while the mRNA levels of IκB-α, P38, and IL-1β were decreased significantly (P ≤ 0.05). On d 43, IMCS significantly upregulated the mRNA levels of TRPM8, IFN- γ, IL-4, IL-6, IL-10, and the protein levels of IκB-α, P38, and the levels of iNOS, TNF-α, IL6 and IL10 in the serum (P ≤ 0.05); whereas it significantly downregulated CIRP, JNK, P38, iNOS, TNF-α mRNA levels, and CIRP, P65, ERK, JNK, IL1β and iNOS protein levels (P ≤ 0.05). Therefore, IMCS can enhance broiler immune function through co-regulation of CIRP and TRPM8 on the NF-κB and MAPK pathways, which facilitate the cold adaptation in broilers., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)