Allyl isothiocyanate (AITC) is a natural product used as a food additive. Due to its strong volatility and broad biological activity, AITC is being considered as a bio-fumigant to control soil-borne fungal diseases in agriculture, creating an urgent need for evaluation of the antifungal activity of AITC. Here, AITC-trigged growth inhibition, sensing, and related molecular mechanisms were analyzed in Fusarium solani by employing morphological, genetic, and transcriptional profiling analysis. The results indicated that AITC causes rapid inhibition of F. solani after 5 min, hyphal deformity, and electrolyte leakage. A yeast-like vacuolar transient receptor potential channel regulator (FsYvc1, a STRPC family member) was identified in F. solani that is play a switch role for sensing AITC. Genetic evidence suggests the gene FsYvc1 involved in growth, development, and pathogenicity. Loss of FsYvc1 resulted in hypersensitivity of F. solani to AITC and high levels of reactive oxygen species with weak responses to CaCl2, NaCl, KCl, SDS, and Congo red as well as triadimefon, triadimenol, chlorothalonil, and azoxystrobin. Transcription profiling and qRT-PCR showed that three transcripts were up-regulated in the wild type (WT) but down-regulated in the FsYvc1 mutants (?FsYvc1), which were involved in TRP ion channel expression, biosynthetic process, and oxidoreductase activity. Three transcripts down-regulated in WT but up-regulated in the ?FsYvc1 were involved in metabolic process, proper enzyme folding (GrpB protein), and the response to abiotic stress (WD40 repeat). This study provides novel insights into the toxicity mechanisms of AITC. Considering the key role of FsYvc1, it could act as a new molecular target for future fungicide development. Overall design: Total RNA from the 12 samples, including WT (F. solani) and ?FsYvc1-17 that were treated and untreated with or without AITC at 4.8 µg/mL for 3 hours, with three biological replicates.