Fruits are a major source of nutrition in human diets, providing carbohydrates, fiber, vitamins and phytonutrients. Carotenoids are a principal class of compounds found in many fruits, providing nutritional benefits both as precursors to essential vitamins and as antioxidants. Molecular characterization revealed that the tomato high pigment mutant genes (hp1 and hp2) encode UV-DAMAGED DNA BINDING PROTEIN-1 (DDB1) and DE-ETIOLATED-1 (DET1) homologs, respectively, and both are essential components of the recently identified CUL4-based E3 ligase complex. Here we have isolated a tomato CUL4 homolog and performed yeast two-hybrid assays to suggest possible association of tomato DDB1 with CUL4 and DET1. Real-time RT-PCR analysis indicated that both HP1 and CUL4 are expressed constitutively. Abscisic acid is implicated in plastid division control and its application substantially enhances HP1/DDB1 mRNA accumulation. Transformation of constructs expressing CUL4-YFP and DDB1-YFP fusion proteins driven by the CaMV 35S promoter reveals that both CUL4 and DDB1 are targeted to tomato plastids and nuclei simultaneously. Using fruit-specific promoters combined with RNAi technology, we show that downregulated DDB1 expression in transgenic fruits results in a significant increase in the number of plastids and corresponding enhanced pigment accumulation. CUL4-RNAi repression lines provide insight regarding CUL4 function during tomato development, and reveal that this tomato cullin is important in the regulation of plastid number and pigmentation, which in turn have a direct impact on fruit nutrient quality.