Objective: To construct two shRNA lentiviral vectors targeting NSD2 (nuclear receptor binding SET domain protein 2) gene and establish a triple-negative breast cancer cell line with stable knockdown of NSD2. Methods: Two shRNA sequences targeting NSD2 gene (shNSD2-1# and shNSD2-2#) were designed, synthesized and cloned into a lentiviral vector (pGLV10/U6/RFP/Puro). Positive clones confirmed by DNA sequencing. Subsequently, NSD2 shRNA lentiviral vector and packing plasmids were co-transfected into 293T cells. The titer of virus was detected according to the expression of red fluorescence protein (RFP). MDA-MB-231 cells were transfected with the packaged lentiviral vector, and the stable cell lines with NSD2 knockdown were obtained after puromycin-resistance screening. The mRNA level of NSD2 was detected by real-time PCR. Western blot was performed to detect the protein level of NSD2 and its downstream target, H3K36me2. Results: Two specific lentiviral RNAi vector targeting NSD2 gene were successfully constructed. Compared to the negative group, the mRNA and protein levels of NSD2 were significantly reduced in shNSD2-1# group and shNSD2-2# group. NSD2-catalyzed H3K36me2 was also downregulated. Conclusion: The MDA-MB-231 cell line with stable knockdown of NSD2 is constructed successfully. The knockdown of NSD2 efficiently suppressed its function of methylating H3K36, providing a foundation for further studying the role and mechanism of NSD2 in breast cancer.