Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system, and to lay foundation for the functional study of MATP gene.Methods Specific primers of MATP were designed according to the report in http://crispr.mit.edu/ website. The primers were linked to pCAS9/gRNA1 vector. Then the positive vector was transfected into mouse melanoma B16F10 cells, and monoclonal cell lines were obtained by the infinite dilution method. After the genomes of different monoclonal cell lines were extracted and sequenced, the cell lines with MATP gene cleavage were screened, and the expression of MATP in these cell lines was verified by Western-blot analysis. Results Three MATP gene knockout cell lines were successfully obtained. The western-blot results showed that the cell lines did not express MATP protein. Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.