Acclimation can alleviate the negative impacts of adverse environmental factors on an organism. To investigate the effects of prior cold stimulation on inflammatory and immune regulation in ileum of cold-stressed broilers, 360 1-d-old chicks (Arbor Acres) were divided into 3 groups (G1, G2, G3) and raised at normal (thermal comfort) temperatures during 1 to 7 d. From 8 d, G1 (control) continued to be raised at normal temperatures, whereas G2 and G3 (treatments) were cold-stimulated at 3°C and 12°C, respectively, below the temperature of G1, but not below 17°C (reached at 32 d in G2 and 14 d in G3). At 42 d, all the groups were subjected to a 24-h acute cold stress of 7°C, designated as S1, S2, and S3. Ileum tissues and serum of the birds were collected at 42 d and 43 d to detect the levels of pro-inflammatory and immune-related factors as well as morphology changes. At 42 d, ileum of G1 and G2 had intact morphological structure and clear outline, with no differences in levels of iNOS, NF-κB, COX-2, PTGEs, TNF-α, IFN-γ, or IL-4 (P > 0.05). G3 ileum suffered inflammatory injuries, with levels of iNOS, COX-2, PTGEs, TNF-α, and IL-4 being higher than those of G1 (P < 0.05). At 43 d, compared with G1, S1 had severely damaged ileum, increased levels of iNOS, NF-κB, COX-2, PTGEs, TNF-α, and IL-4 (P < 0.05), but decreased level of IFN-γ (P < 0.05). The morphological structure of S2 was intact, and there were no differences between G2 and S2 in levels of NF-κB, PTGEs, TNF-α, IFN-γ, or IL-4 (P > 0.05). Compared with S1, S2 had decreased levels of iNOS, NF-κB, COX-2, PTGEs, TNF-α, and IL-4 (P < 0.05), but increased level of IFN-γ (P < 0.05). There were no differences between G1 and S2 in levels of NF-κB, PTGEs, TNF-α, IFN-γ, or IL-4 (P > 0.05). The results demonstrate that a 3°C-lower-than-normal temperature stimulation of the broilers from 8 to 42 d led to cold acclimation. This prior cold acclimation effectively alleviated elevation of pro-inflammatory cytokines and Th1/Th2 imbalance of the birds induced by subsequent cold stress.