Virus-induced gene silencing (VIGS) is known as a rapid and efficient system for studying functions of interesting genes in plants. Tobacco rattle virus (TRV) is widely applied for the gene silencing of many plants. Although spinach is a TRV-susceptible plant, a TRV-based VIGS system has not yet been developed for spinach. In this study, we established a TRV-based VIGS system for spinach. To evaluate the functionality of the TRV-based VIGS system, the phytoene desaturase gene (SoPDS) was first isolated from spinach as a marker gene. Then, the VIGS vector pTRV2 was combined with the partial fragment of SoPDS gene in sense or antisense orientation. Using the Agrobacterium infiltration method, we introduced the pTRV2-SoPDS clone to silence the SoPDS gene in spinach. SoPDS was efficiently silenced, and consequently, greater than 90% of newly emerging leaves exhibited severe chlorosis symptoms in the treated plants. Levels of chlorosis symptoms were similar in both plants infected with pTRV2 vectors harboring sense (SoPDS_S) or antisense (SoPDS_A) gene fragments. Quantitative analysis of SoPDS gene expression by qRT-PCR revealed that gene expression was reduced by greater than 90% in both SoPDS_S and SoPDS_A VIGS plants. Chlorosis on leaves was prolonged up to 4~5 wk after Agrobacterium infiltration. The TRV-based VIGS system was effective in silencing the SoPDS gene in spinach, suggesting that it can be a useful reverse genetics tool for the functional study of spinach genes.