Tomato spotted wilt orthotospovirus (TSWV) is a serious plant pathogen of crops that causes serious damage in the agriculture industry. The damage caused by TSWV has made the development of new and effective botanical antiviral agents against the disease increasingly urgent. Many kinds of compounds have been found to control disease caused by TSWV. However, novel alkaloid for the effectively control of this disease remain rarely. In this study, a series of compounds of novel alkaloid were extracted from Corydalis saxicola Bunting and their structure was elucidated by nuclear magnetic resonance mass spectrometry (NMR- MS). The compounds demonstrated anti-TSWV activity in vivo. The protective and curative activities of berberine (ber) (100 μg/mL) were 62.00 % and 50.00 %, respectively, which were significantly higher than those of commercial ningnanmycin and other compounds. Several concentrations of ber were tested in vitro, and the most effective concentration with an anti-TSWV activity greater than 76.00 % (200 μg/mL). Transcriptome analysis revealed that ber induced multiple genes related with glutathione S-transferase activity, auxin-activated signaling pathway, sesquiterpenoid and triterpenoid biosynthesis, intracellular trafficking, secretion, and vesicular transport. In addition, GO, KEGG and RT-qPCR showed that ber primarily acted on the enzyme glutathione S-transferase (GSTAU25) to improve the resistance to TSWV infection. Subsequent virus-induced gene silencing (VIGS) combined with overexpression was used to verify that ber regulated the inhibition of the expression of NSs and NSm genes via GSTAU25. Therefore, the results indicated that ber can stimulate GSTAU25 and thereby enhance the defense against TSWV. This study identified ber as a promising natural botanical antiviral agent for future study, and the results provide a theoretical basis for the application of ber in Orthotospovirus management.