Glycerol-3-phosphate (G3P) is a proposed regulator of plant defense signaling in basal resistance and systemic acquired resistance (SAR). The GLY1-encoded glycerol-3-phosphate dehydrogenase (G3PDH) and GLI1-encoded glycerol kinase (GK) are two key enzymes involved in the G3P biosynthesis in plants. However, their physiological importance in wheat defense against pathogens remains unclear. In this study, quantification analysis revealed that G3P levels were significantly induced in wheat leaves challenged by the avirulent Puccinia striiformis f. sp. tritici (Pst) race CYR23. The transcriptional levels of TaGLY1 and TaGLI1 were likewise significantly induced by avirulent Pst infection. Furthermore, knocking down TaGLY1 and TaGLI1 individually or simultaneously with barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) inhibited G3P accumulation and compromised the resistance in the wheat cultivar Suwon 11, whereas the accumulation of salicylic acid (SA) and the expression of the SA-induced marker gene TaPR1 in plant leaves were altered significantly after gene silencing. These results suggested that G3P contributes to wheat systemic acquired resistance (SAR) against stripe rust, and provided evidence that the G3P function as a signaling molecule is conserved in dicots and monocots. Meanwhile, the simultaneous co-silencing of multiple genes by the VIGS system proved to be a powerful tool for multi-gene functional analysis in plants.