Environmental stresses can regulate gene expression patterns by modulating histone activity. However, the effect of low-temperature stress on whole-genome histone modification in fish has not been reported. To explore the effect of low-temperature stress on histone modification in zebrafish () cells, the experimental conditions for chromatin immunoprecipitation (ChIP) were established and optimized. The optimum NP-40 concentration for lysing cells was 0.2%. The optimal time of sonication with a Covaris S220 to break the chromatin was 20 min. Primers targeting the -actin promoter region were used to perform routine PCR to verify the preliminary ChIP experiment results. Optimized experimental conditions resulted in a very low background, with nearly no IgG band in the agarose gel. Moreover, the lack of H3K27me3 in the -actin promoter region, accompanied by enriched H3K4me3 and H3K27ac, was observed as predicted. Zebrafish embryonic ZF4 fibroblasts cultured under normal condition (28℃) and cold-acclimated ZF4 cells under long-term cold exposure (18℃ for 30 days) were evaluated with the optimized ChIP protocol. The effect of cold acclimation on the expression of tumor necrosis factor tnfb promoter was detected by qPCR and ChIP-qPCR, respectively. The qPCR and ChIP-qPCR results showed that was upregulated after cold acclimation and that H3K4me3 and H3K27ac were enriched in the promoter region of , while enrichment of H3K27me3 showed no significant change. This suggests that cold pressure regulates expression by affecting H3K4me3 and H3K27ac levels in the promoter region of . In conclusion, the established and optimized ChIP method can be used to study histone modification in ZF4 cells and provides a foundation for further analysis of the effect of low-temperature stress on whole-genome histone modification in zebrafish cells.