Abstarct: Fusarium solani f. Sp.cucurbitae is the causal agent of root, crown and fruit rot of Cucurbitaceae and was considered as an economic problem for growers in recent years. In this study, we aimed to isolate and identification of Fusarium solani f. Sp. Cucurbitae causes root, crown and fruit rot of Cucurbitaceae and study of the molecular and enzymatic response of susceptible (Negin) and resistance (Khasib) cultivars of cucumber to this pathogen. Result revealed that F. Solani f. Sp.cucurbitae is dominant species and race 1 is the dominant race in studied region. Analysis of gene exxpression data revealed that level of pr-1 exxpression in control plants of resistance cultivar is more than that of susceptible cultivar (123.5, 4.11 and seven times higher at 3, 7 and 10 dpi days respectively), whereas Phenylalanine ammonia-lyase (pal) gene was more up-regulated in susceptible cultivar than resistance cultivar (1.19, 64.15 and 1.14 times higher at 3, 7 and 10 dpi respectively). Ten days after inoculation of fungi, pr1 and pal were upregulated compared to control plants in Khasib cultivar (1.5 and 12.28 times higher for pal and pr1, respectively). Pretreatment of plants by salicylic acid causes increase in the level of pal and pr1 transcxripts as compared to inoculated plant by fungi, so that in Khasib cultivar up-regulated dramatically three days after inoculation (22.12 times higher for pr1 and 53.66 times higher for pal) and in Negin cultivar they were up-regulated dramatically ten days after inoculation (53.5 times higher for pr1 and 173 times higher for pal). It is demonstrated that catalase activity was increased in Khasib and Negin, three and seven days after fungi inoculation (4.05 and four times higher in Khasin and 2.33 and 1.9 times higher in Negin as compared to control plants). The salicylic acid treatment causes increase in enzyme activity in Khasib cultivar especially at 3 dpi day (2.85 times higher than control plants) and phosphite potassium (20 mm) treatment increased catalase activity in Negin cultivar especially at 3 dpi (3.4 times higher than control plants). The activity of pal enzyme had no significant changes in Negin cultivar after treatments and inoculation of fungi, whereas it was activated significantly after some of treatments and inoculation of fungi only at 7 dpi especially with pretreatment of the plant by salicylic acid (2.49 times higher than control plants). Results revealed that salicylic acid (2 mm) and phosphite potassium (20 mm and 36 mm) increase defense genes exxpression and activity of the related enzyme to biotic stresses and subsequently increase resistance to F. Solani f. Sp. Cucurbitae.