Cycloleucine, a non-proteinogenic amino acid, has been identified as a potent inhibitor of fungal growth; however, its molecular mechanisms of action against the edible mushroom Sparassis latifolia remain inadequately understood. In this study, we integrated transcriptomic and m6A methylome analyses to elucidate the inhibitory effects of cycloleucine on S. latifolia. Our findings demonstrate that cycloleucine treatment suppresses hyphal growth and biomass accumulation in S. latifolia, which are associated with a significant reduction in global RNA m6A levels. Through meRIP-seq analysis, we identified 67 differentially m6A-modified genes, including aif1 (apoptosis-inducing factor 1) and mfs1 (major facilitator superfamily transporter 1), suggesting their involvement in growth regulation. Genome-wide screening and expression detection revealed that EVM0008471-mediated m6A demethylation plays a potential role in controlling hyphal growth dynamics. This study provides the first evidence of m6A-dependent growth regulation in S. latifolia, advancing the understanding of fungal epitranscriptomics and offering potential targets for optimizing industrial mushroom cultivation.