Abstract: Diacylglycerol acyltransferase (DGAT) is a key enzyme in the synthesis pathway of triglyceride (TAG), which plays an important role in plant growth, development and stress response. In this study, the NtDGAT3 gene was cloned from tobacco cultivar K326. The expressions of NtDGAT3 were analyzed by qRT-PCR in different tissues of tobacco, and in plants under cold stress. Meanwhile, the NtDGAT3 loss-of-function mutant plants were obtained by CRISPR/Cas9 technology to evaluate low-temperature tolerance. The results showed that the NtDGAT3 gene was expressed in roots, stems, leaves, and the highest expression was found in leaves. The expression level of NtDGAT3 was induced under cold stress, which indicated that NtDGAT3 might be involved in tobacco cold stress response. The results showed that knockout mutation of ntdgat3 was sensitive to cold stress. At low temperature, the ion leakage rate of ntdgat3 knockout plant leaves was significantly higher than that of wild-type tobacco, and the increase in malondialdehyde (MDA) content was much higher than that of wild-type tobacco. The expression level of PLDα1, a key gene of membrane lipid metabolism, was significantly higher than that of the wild type, while the expression level of PLDδ was significantly lower than that of the wild type. These results suggest that NtDGAT3 is involved in tobacco response to cold stress by regulating the expression of PLDα1 and PLDδ.