Belonging to the Brazilian flora, the species Hancornia speciosa (Gomes), known as mangabeira, has bioactive compounds of interest, such as flavonoids, xanthones, and proanthocyanidins. The objective of this study was to determine how the supplementation of sugars in culture medium affects the osmotic potential of the medium, as well as its influence on cell growth and on the concentration of phenolic compounds. For this purpose, after 90 days of subculture, 20 ml aliquots of the cultures were added to flasks containing 20 ml of medium with different sugars (glucose, fructose, sucrose, mannitol, and sorbitol) under a 16-h photoperiod with a spectral range between 400 and 700 nm of photosynthetically active radiation (45-55 μmol m -2 s -1 ) in a shaker at 110 rpm. After 30 days, the pH, electrical conductivity, osmotic potential, biomass accumulation, and concentrations of phenolic compounds were evaluated. Regardless of their concentration in the medium, the sugars sorbitol and mannitol provided more unfavorable conditions for water absorption at the cellular level, reducing the water potential of the medium. Sucrose favored greater water absorption and biomass accumulation. Among the various sugar concentrations, 3% (30 g/L) sucrose or glucose improved the accumulation of fresh and dry cell weight and the production of polyphenols such as chlorogenic acid, epicatechin, rosmarinic acid, hesperidin, rutin, and quercetin. In addition, they resulted in a higher osmotic potential of the medium and larger cells than other carbon sources. Despite the differences in cell size, no culture conditions compromised cell survival.