Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme that regulates the gluconeogenesis pathway in plants. While the biochemical properties of PEPCK have been reported for many species, its physiological function is not fully understood in plants with fresh berry-type fruit. To clarify its physiological role(s) in the tomato plant, the effect of excessive PEPCK was investigated using transgenic lines overexpressing SlPEPCK by either the CaMV 35S constitutive promoter or the fruit-specific E8 promoter. Detailed characterization of the phenotypic and metabolic properties of the 35S promoter-driven lines revealed that the transgenic seedlings exhibited earlier germination and better seedling growth compared with the wild type. Interestingly, seedling growth at 10 days after sowing of the transgenic lines was enhanced by an exogenous sucrose supply. These results suggest that PEPCK enhances seedling growth through PEPCK/pyruvate kinase-mediated pathway rather than gluconeogenesis during germination. In addition, increased soluble sugars and decreased malate contents were observed in red-ripe fruit in both the 35S and E8 promoter-driven lines, indicating the participation of gluconeogenesis in sugar/acid metabolism during fruit ripening. The present results are totally opposite to those observed in PEPCK-suppressed RNAi lines, which were investigated in our previous work. The results indicate the regulatory role of PEPCK in post-germination growth and sugar/organic acid accumulation in ripening tomato fruit.