Aims Fusarium oxysporum is a causal disease that threatens watermelon production, but little information on the molecular mechanisms involved in host defense is available. To understand the defense response, a proteome-level changes that occur in watermelon roots during F. oxysporum infection were investigated. Methods We utilized two-dimensional gel electrophoresis (2-DE) to compare changes in the root proteome profiles and validated their expression using real-time PCR. Results A total of 690 spots were detected, and 32 proteins had significant changes in abundance and were further identified by mass spectrometry. These proteins were mainly involved in metabolism, stress and defense and amino acid biosynthesis. RT-PCR analysis revealed that transcripts corresponding to the nine randomly selected proteins could be significantly induced, their expression patterns were consistent with the proteomic results except for Apx and Tdh. The involvement of these proteins in regulating watermelon response against F. oxysporum is discussed. Conclusions The reprogrammed proteins were involved in several biological processes, which indicates that watermelon can directly alter the abundance of these proteins to establish a defense response. This work helps us understand the basic processes during the watermelon-F. oxysporum interaction and may contribute to improve resistance breeding toward this pathogen.