Insufficient leaf photoassimilation could allow mobilized carbohydrate reserves to contribute to berry sugar accumulation. However, the extent of this contribution during rapid and slow berry sugar accumulation is undefined. The potential effect of leaf-to-fruit ratio and water availability on carbohydrate reserve distribution in potted Tempranillo grapevines was examined during berry maturation. Within each leaf-to-fruit ratio treatment (full and 50% leaves), vines were grown under full or 50% reduced irrigation regimes. Dry biomass development, and the starch and soluble sugar concentrations were determined in the roots, trunks, stems and leaves. Berry sugar and anthocyanin accumulation were also assessed. Under full irrigation, no starch remobilization from roots was observed, regardless of the leaf-to-fruit ratio. Under reduced water supply, starch remobilization from roots was concurrent with rapid berry sugar accumulation, especially in grapevines with low leaf-to-fruit ratio. Soluble sugar accumulation coincided with starch depletion in the roots of grapevines under reduced water availability. When berry sugar accumulation slowed, an increase in carbohydrates was observed in the roots. Sustained water constraints during rapid berry sugar accumulation resulted in a forced reliance on stored carbohydrates to support berry sugar accumulation, but did not significantly alter the tempo of berry sugar and anthocyanin accumulation. A reduced leaf-to-fruit ratio intensified the reliance of fruit sugar accumulation on stored carbohydrates. Besides the importance of post-harvest carbohydrate reserve replenishment when root carbohydrate reserves are depleted during berry maturation, the reserves are also refilled during maturation when berry sugar accumulation slows. This study showed distinctly that root carbohydrate replenishment could already start a few weeks before harvest, and this replenishment could be important when the post-harvest carbon assimilation period is ineffective.