Abstract: In order to study the expression pattern and function of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in tobacco, NtDXR gene was cloned from tobacco cultivar Hongda (Nicotiana tabacum) and the expression level of NtDXR was also analysed in different tissues and organs. The CRISPR/Cas9 plant expression vector was constructed and the NtDXR knockout mutations were obtained. The results showed that NtDXR had the highest expression level in flower, higher in pistil and leaves. DNA insertion, deletion and displacement had been found in the target sites of NtDXR by targeted mutagenesis using the type II CRISPR/ Cas9 systemClustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9. Partial T₀ mutant plants showed obviously photo-bleaching phenotypes, which implied that NtDXR played an important role in chlorophyll synthesis, and it suggested that the biosynthesis of plastid pigment in tobacco might be blocked by the disfunction of NtDXR.