Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography-mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.