MicroRNAs (miRNAs) play pivotal roles in various biological processes across kingdoms. Many plant miRNAs have been experimentally identified or predicted by bioinformatics mining of small RNA databases. However, the functions of these miRNAs remain largely unknown due to the lack of effective genetic tools. Here, we report a virus-based microRNA silencing (VbMS) system that can be used for functional analysis of plant miRNAs. VbMS is performed through tobacco rattle virus-based expression of miRNA target mimics to silence endogenous miRNAs. VbMS of either miR172 or miR165/166 caused developmental defects in Nicotiana benthamiana. VbMS of miR319 reduced the complexity of tomato (Solanum lycopersicum) compound leaves. These results demonstrate that tobacco rattle virus-based VbMS is a powerful tool to silence endogenous miRNAs and to dissect their functions in different plant species.