On the basis of our previous studies on the antiviral mechanism against tobacco mosaic virus (TMV) and structure-activity relationship of phenanthroindolizidine alkaloids, a series of 9-substituted tylophorine derivatives targeting TMV RNA were designed, synthesized, and assessed for their anti-TMV activities. The bioassay results indicated that most of these compounds showed good in vivo anti-TMV activities, and some of them displayed higher activity than that of commercial ribavirin. Especially, the anti-TMV activities of compound 3b, 4, and 6 are 2-3 times higher than that of commercial ribavirin, according to EC50 values. In this work, we have demonstrated an effective way to design new inhibitors against plant virus and developed 9-ethoxy methyl tylophorine (4) with excellent anti-TMV activity (in vitro activity, 70.2%/500 μg/mL and 27.1%/100 μg/mL; inactivation activity, 67.7%/500 μg/mL and 30.5%/100 μg/mL; curative activity, 65.3%/500 μg/mL and 30.8%/100 μg/mL; and protection activity, 65.9%/500 μg/mL and 36.0%/100 μg/mL) as a potential plant viral inhibitor. © 2021 American Chemical Society.