Background: Prospective studies indicate that tomato consumers are protected against prostate cancer. Lycopene has been hypothesized to be responsible for tomato health benefits. Objectives: Our aim was to differentiate the effects of tomato matrix from those of lycopene by using lycopene-rich red tomatoes, lycopene-free yellow tomatoes, and purified lycopene. Design: Thirty healthy men (aged 50–70 y old) were randomly assigned to 2 groups after a 2-wk washout period. In a crossover design, each group consumed yellow and red tomato paste (200 g/d, which provided 0 and 16 mg lycopene, respectively) as part of their regular diet for 1 wk separated by 2 wk of washout. Then, in a parallel design, the first group underwent supplementation with purified lycopene (16 mg/d) for 1 wk, whereas the second group received a placebo. Sera collected before and after the interventions were incubated with lymph node cancer prostate cells to measure the expression of 45 target genes. Results: Circulating lycopene concentration increased only after consumption of red tomato paste and purified lycopene. Lipid profile, antioxidant status, prostate-specific antigen, and insulin-like growth factor I were not modified by consumption of tomato pastes and lycopene. We observed significant up-regulation of IGFBP-3 and Bax:Bcl-2 ratio and down-regulation of cyclin-D1, p53, and Nrf-2 after cell incubation with sera from men who consumed red tomato paste when compared with sera collected after the first washout period, with intermediate values for yellow tomato paste consumption. Cell incubation with sera from men who consumed purified lycopene led to significant up-regulation of IGFBP-3, c-fos, and uPAR compared with sera collected after placebo consumption. Conclusion: Dietary lycopene can affect gene expression whether or not it is included in its food matrix.