Free amino acids (AAs) formed in fermented meat products are important nitrogen sources for the survival and metabolism of contaminating fungi. These AAs are mainly regulated by the TORC1-Tap42signaling pathway.Fusariumspp., a common contaminant of fermented products, is a potential threat to food safety. Therefore, there is an urgent need to clarify the effect of different AAs onFusariumspp. growth and metabolism. This study investigated the effect of 18 AAs onFusarium oxysporum(Fo17) growth, sporulation, T-2 toxin (T-2) synthesis andTri5expression throughTap42gene regulation. Co-immunoprecipitation and Q Exactive LC-MS/MS methods were used to detect the interacting protein of Tap42 during specific AA treatment.Tap42positively regulated L-His, L-Ile and L-Tyr absorption for Fo17 colony growth. Acidic (L-Asp, L-Glu) and sulfur-containing (L-Cys, L-Met) AAs significantly inhibited the Fo17 growth which was not regulated byTap42. The L-Ile and L-Pro addition significantly activated the sporulation of ΔFoTap42.L-His and L-Ser inhibited the sporulation of ΔFoTap42. In T-2 synthesis, ΔFoTap42was increased in GYM medium, but was markedly inhibited in L-Asp and L-Glu addition groups. Dose–response experiments showed that 10–70 mg/mL of neutral AA (L-Thr) and alkaline AA (L-His) significantly increased the T-2 production andTri5expression of Fo17, butTri5expression was not activated in ΔFoTap42. Inhibition of T-2 synthesis andTri5expression were observed in Fo17 following the addition of 30–70 mg/mL L-Asp. KEGG enrichment pathway analysis demonstrated that interacting proteins of Tap42 were from glycerophospholipid metabolism, pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, glycolysis and gluconeogenesis, and were related to the MAPK and Hippo signaling pathways. This study enhanced our understanding of AA regulation in fermented foods and its effect onFusariumgrowth and metabolism, and provided insight into potential ways to control fungal contamination in high-protein fermented foods.