The cultivation of spinach (Spinacia oleracea L.) has been increasing during the last years in controlled environment agriculture, where light represents a key factor for controlling plant growth and development and the highest energetic costs. The aim of the experiment was to evaluate the plant’s response to two light intensities, corresponding to an optimal and a reduced level, in terms of the photosynthetic process, photoassimilates partitioning, and the biosynthesis of sucrose and starch. Plants of spinach cv. ‘Gigante d’Inverno’ were grown in a phytotron under controlled conditions, comparing two values of photosynthetic photon flux density (PPFD), 800 μmol m−2 s−1 (800 PPFD) and 200 μmol m−2 s−1 (200 PPFD), at a 10 h light/14 h dark regime. Compared to 800 PPFD, under 200 PPFD, plants showed a reduction in biomass accumulation and a redirection of photoassimilates to leaves, determining a leaf expansion to optimize the light interception, without changes in the photosynthetic process. A shift in carbon partitioning favouring the synthesis of starch, causing an increase in the starch/sucrose ratio at the end of light period, occurred in low-light leaves. The activity of enzymes cFBAse, SPS, and AGPase, involved in the synthesis of sucrose and starch in leaves, decreased under lower light intensity, explaining the rate of accumulation of photoassimilates.