Grafting can improve the resistance of watermelon to soil-borne diseases. However, the molecular mechanism of defense response is not completely understood. Herein, we used a proteomic approach to investigate the molecular basis involved in grafted watermelon leaf defense against f.sp. () infection. The bottle gourd rootstock-grafted (RG) watermelon seedlings were highly resistant to compared with self-grafted (SG) watermelon plants, with a disease incidence of 3.4 and 89%, respectively. Meanwhile, grafting significantly induced the activity of pathogenesis-related proteases under challenge. Proteins extracted from leaves of RG and SG under inoculation were analyzed using two-dimensional gel electrophoresis. Thirty-nine differentially accumulated proteins (DAPs) were identified and classified into 10 functional groups. Accordingly, protein biosynthetic and stress- and defense-related proteins play crucial roles in the enhancement of disease resistance of RG watermelon seedlings, compared with that of SG watermelon seedlings. Proteins involved in signal transduction positively regulated the defense process. Carbohydrate and energy metabolism and photosystem contributed to energy production in RG watermelon seedlings under infection. The disease resistance of RG watermelon seedlings may also be related to the improved scavenging capacity of reactive oxygen species (ROS). The expression profile of 10 randomly selected proteins was measured using quantitative real-time PCR, among which, 7 was consistent with the results of the proteomic analysis. The functional implications of these proteins in regulating grafted watermelon response against are discussed.