Fusarium oxysporum f. sp. cubense (Foc) is the fungal pathogen that causes Panama disease. Foc is divided into four distinct races depending on host specificity. Foc race 1 (R1) is known to decimate the well-known ‘Gros Michel’ banana cultivar, whereas Cavendish bananas resistant to R1 have been replaced for commercial production. However, the emergence of a hypervirulent strain, tropical race 4 (TR4) has raised serious concerns for Cavendish varieties. To reveal the virulence mechanism of Foc TR4 and its interaction with hosts, we conducted a dual transcriptome study in the Cavendish susceptible Pei-Chiao (PC) variety and somaclonal-derived resistant Tai-Chiao No.7 (TC7) variety. Among the differentially expressed candidate genes, we identified a putative Foc13813 gene that shares homologues only present in Fusarium species complex. Foc13813 gene exhibited elevated expression during the late stage of infection in PC but not detectable in TC7, consistently validated by RT-qPCR. To functionally characterize its role in Foc TR4, gene deletion and complementation strains were created. The deletion mutants showed significant impairment of virulence in PC and TC7. Histological section revealed that deletion mutants failed to infect feeder roots, rhizome and pseudostem tissues compared to wild-type strain. This study reports the identification of a novel gene required for pathogenicity of Foc TR4 and current investigation aims to reveal the mechanisms of Foc TR4 toward Cavendish bananas.