In this study, a wheat defensin gene TaPDF35 was cloned from sharp eyespot-resistant wheat cultivar CI12633, and its expression and function were analyzed. TaPDF35 contains an open reading frame (ORF) with 249 bp-length, and encodes a peptide TaPDF35 consisting of 82 amino acid. The peptide TaPDF35 includes a signal peptide with 27 amino acid-length at its N-terminus. The mature protein forms a typical αβ (CSαβ) motif, which contains an α helix and a triple-stranded anti-parallel β-sheet and is stabilized through four disulfide bridges formed by eight cysteines. Real-time RT-PCR analysis showed that the expression levels of TaPDF35 gene in sharp eyespot-resistant wheat cultivars CI12633 and Shanhongmai were significantly higher than those in sharp eyespot-susceptible wheat cultivars Yangmai158 and Wenmai 6. The expression of TaPDF35 was up-regulated after infection of Rhizoctonia cerealis (the pathogen of sharp eyespot). Through BSMV-based virus-induced gene-silencing (VIGS) method, TaPDF35 silencing CI12633 plants induced by BSMV: TaPDF35 showed more susceptible to Rhizoctonia cerealis infection compared with CI12633 control plants induced by BSMV:GFP. The results suggest that the expression of TaPDF35 is required for wheat defense response to Rhizoctonia cerealis infection.