F-box proteins function in the recruitment of proteins for SCF ubiquitination and proteasome degradation. Here, we studied the role of Fbp1, a nonessential F-box protein of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. The Δfbp1 mutant showed a significant delay in the production of wilt symptoms on tomato plants and was impaired in invasive growth on cellophane membranes and on living plant tissue. To search for target proteins recruited by Fbp1, a combination of sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) was used to compare proteins in mycelia of the wild-type and Δfbp1 mutant. The proteomic approach identified 41 proteins differing significantly in abundance between the two strains, 17 of which were more abundant in the Δfbp1 mutant, suggesting a possible regulation by proteasome degradation. Interestingly, several of the identified proteins were related to vesicle trafficking. Microscopic analysis revealed an impairment of the Δfbp1 strain in directional growth and in the structure of the Spitzenkörper, suggesting a role of Fbp1 in hyphal orientation. Our results indicate that Fbp1 regulates protein turnover and pathogenicity in F. oxysporum.