A soil-inhabiting fungus, Fusarium oxysporum f. sp. cucumerinum (Foc), is a causal agent of cucumber Fusarium wilt, one of the most serious diseases in cucumber cultivars. We previously found that the virulence of Foc significantly enhanced after serially passaged through resistant cucumber cultivar. To better understand its virulence mechanism, a low virulence strain foc-3b and its virulence-increased variant Ra-4 obtained from serial passage were selected and their transcriptome profiles in infected cucumber roots at 24 and 120 h after inoculation (hai) were analyzed. A series of differentially expressed genes (DEGs) potentially involved in fungal pathogenicity and pathogenicity variation were identified.