Plant microRNAs (miRNAs) play pivotal roles in many biological processes. Although many miRNAs have been identified in various plant species, the functions of these miRNAs remain largely unknown due to the shortage of effective genetic tools to block their functional activity. Recently, miRNA target mimic (TM) technologies have been applied to perturb the activity of specific endogenous miRNA or miRNA families. We previously reported that Tobacco rattle virus (TRV)-based TM expression can successfully mediate virus-based miRNA silencing/suppression (VbMS) in plants. In this study, we show the Potato virus X (PVX)-based TM expression causes strong miRNA silencing in Nicotiana benthamiana. The PVX-based expression of short tandem target mimic (STTMs) against miR165/166 and 159 caused the corresponding phenotype in all infected plants. Thus, a PVX-based VbMS is a powerful method to study miRNA function and may be useful for high-throughput investigation of miRNA function in N. benthamiana.