Abstract: Low water temperature greatly influences the physiology and development of toleost fish. The fluidity and stability of biofilm in fish will be greatly reduced at low temperature, which will affect the physiological process of fish, including nutrient metabolism and immune physiological response. Fatty acid composition plays an important role in maintaining membrane fluidity and energy metabolism under low temperature stress. Fatty acid elongase is a key rate limiting enzyme in the process of fatty acid elongation in organisms, which is very important to improve the ability of fish to resist cold stress. Fatty acid elongase 8 (elovl8) is a fatty acid elongating enzyme gene identified in teleost fish, but its role in low water temperature resistance of fish remains unclear. This study was aimed at investigating the role of elovl8 in cold stress resistance of zebrafish, and providing a theoretical basis for improving the mechanism of cold stress resistance of zebrafish. In this study, we used CRISPR/cas9 technology to construct zebrafish elovl8a^-/-, elovl8b^-/-, and DKO knockout models, and explored the effect of elovl8 deletion on cold stress resistance of zebrafish through histological observation, Quantitative Real-time PCR (qPCR), fatty acid composition and other experimental methods. The results showed that the loss of elovl8 significantly reduced the survival rate of zebrafish in low water temperature environment accompanied by obvious lipid deposition and liver fatty lesions. The liver sections showed obvious vacuoles in the liver of elovl8a^-/- and elovl8b^-/- zebrafish under cold stress. The results of qPCR showed that the deletion of elovl8a and elovl8b resulted in significant changes in genes related to lipid metabolism: the transcription levels of stearoyl CoA desaturase (scd) and acetyl-CoA carboxylase a (acaca) in elovl8a^-/- liver increased significantly, and the lipolysis genes included patatin-like phospholipase domain containing 2 (pnpla2), activated hormone sensitive a (lipea) and lipoprotein lipase (lpl) expression level decreased significantly. The expression levels of fasn, scd, acaca, pnpla2, lipea and lpl in elovl8b^-/- liver increased significantly, while the expression levels of fasn, scd, acaca, lipea and lpl in DKO liver decreased significantly, only the expression level of pnpla2 increased. The different changes of lipid metabolism expression levels in elovl8a^-/-, elovl8b^-/- and DKO zebrafish indicated that the function of elovl8a and elovl8b probably existed difference. Fatty acid composition analysis showed that the loss of elovl8 caused significant accumulation of liver C20:0 fatty acids under low water temperature, suggesting that elovl8 may play a role in the elongation of C20:0. The results of this study showed that elovl8 was closely related to cold tolerance of zebrafish. The absence of elovl8 would weaken the ability of zebrafish to endure low temperature and decrease the survival rate significantly. The lipid metabolism of zebrafish in elovl8a^-/-, elovl8b^-/- and DKO was disturbed under cold stress, resulting in the changes of lipid deposition and fatty acid composition, aggravating the death of zebrafish. The absence of elovl8a and elovl8b resulted in the accumulation of C20:0 content and the inhibition of C22:0 synthesis in zebrafish under low temperature stress, suggesting that elovl8 may be involved in the prolongation of C20:0 fatty acids. This study indicates that elovl8 play an important role in the process of cold stress resistance of zebrafish by regulating lipids metabolism, which provide a new idea for the study on the mechanism of fish cold adaptation.