Hyperhydricity is a physiological disorder impacting plant growth and multiplication and acclimatization of regenerated plantlets. We report the use of calcium nitrate for reversion and acclimatization of banana ‘Grand Naine’ hyperhydric shoots cultured on Murashige and Skoog medium containing agar or gellan. Although 100% rooting of hyperhydric shoots occurred at all concentrations of calcium nitrate, only 50% rooting was recorded in the absence of calcium nitrate. Electrolyte leakage decreased significantly in the reverted banana tissues compared with the hyperhydric tissues. Histochemical staining for reactive oxygen species indicated that reverted banana tissues possess lower levels of both hydrogen peroxide (H2O2) and superoxide (O2 -) than do hyperhydric tissues. Rooting, growth, and survival of the reverted banana plantlets were significantly influenced by calcium nitrate concentrations as well as the type of gelling agent. Reverted banana plantlets in medium containing calcium nitrate (0.5–1 g·L−1) were acclimatized with 100% survival in a growing substrate of peatmoss and vermiculite (1:1).