The aim of this study was to investigate the effect of Moringa oleifera seed isothiocyanate (MITC) on the growth of skin squamous cell carcinoma cells (A431 cells). Following the treatment with different concentrations of MITC, the proliferation, apoptosis and cell cycle changes of A431 cells were detected by the MTT assay, clone formation experiment and flow cytometry; The xenograft tumor model was used to detect the effect of MITC on the growth of A431 cells in vivo. The results showed that the viability of A431 cells decreased gradually with the increase of MITC concentrations and processing time, and after a 72 h stimulation with MITC at 12 μM, the cells survival rate decreased to 10% (p<0.001); The results of the clonal formation experiments showed that MITC (5 and 10 μM) significantly inhibited the clonal formation of A431 cells, and the clonal formation rate after a 24 h stimulation with MITC decreased from 100% to 63.22% (p<0.001) and 26.07% (p<0.001); The results of apoptosis and cell cycle examinations showed that after a 48 h treatment with MITC, the apoptosis rate of A431 cells increased from 17.22% to 31.73% (p<0.001) and 44.77% (p<0.001), and the S-phase cells increased significantly from 7.43% to 14.44% (p<0.001) and 17.43% (p<0.001). The in vivo experiments showed that, after 20 days of MITC intervention in the mice with xenograft tumor, the tumor volume of the mice decreased from 1549.02 mm3 to 857.77 mm3 (p<0.05); The tumor weight decreased from 1.30 g to 0.91 g (p<0.05). The above results show that MITC can inhibit the growth of A431 cells in vitro and in vivo.