Fusarium graminearum is the primary causal agent of Fusarium Head Blight (FHB), a devastating fungal disease on wheat, barley, and other grains. During infection, F. graminearum produces trichothecenes, predominately deoxynivalenol (DON), which contaminates grain and reduces grain yield and quality. Although DON functions as a virulence factor to promote F. graminearum spread in the wheat head, it is not essential for initial infection. When fungal pathogens, such as F. graminearum, infect a host plant, they secrete hundreds of small proteins called effectors that interfere with the plant immune system to promote infection and disease. A recent study identified approximately 150 putative effector-encoding genes that are conserved in F. graminearum species. Gene expression analysis of selected candidates from F. graminearum PH-1 identified 12 effector genes that were highly induced with various expression profiles in wheat heads over a seven-day infection period. To elucidate their role in FHB, deletion mutants were generated for ten effector genes and FHB virulence assays were conducted with dip inoculations. FHB virulence assays showed that deletion mutants of a rhamnogalacturonan acetylesterase homolog, FGSG_04848 (FgRGAE), significantly reduced initial infection and DON in wheat and barley spikes compared to wild-type controls. Replacing the FgRGAE^Δ::Hyg construct with a FgRGAE^WT::Gen construct at the native locus restored FHB disease to WT levels in both wheat and barley heads. In silico analysis of FgRGAE suggests that this gene encodes a putative rhamnogalacturonan acetylesterase, a member of the SGNH-hydrolase family predicted to deacetylate pectin subunits. General esterase activity was experimentally shown using the colorimetric substrate 4-nitrophenyl acetate. FgRGAE may serve as an ideal target to reduce FHB and mycotoxin contamination.