Abstract: Background and Objectives: The efficiency of in vitro culture depends on the composition of the medium used. Micronutrients are essential for all stages of plant development performing a key function in the activation of several enzymes. This study focused on studying the influence of increased copper content in the Murashige and Skoog medium on shoot development, chlorophyll content and rooting response of in vitro cultured banana plants (Musa acuminata L.) cultivar (Grand Nain). Materials and Methods: Banana shoot explants were cultured on shoot multiplication media supplemented with various concentrations (0-8 mg L–1) of copper sulphate (CuSO4) and the shoot growth responses, number of shoots per explant, shoot length, leaf surface area and leaf chlorophyll content were noted 4 weeks after culture. The effect of applied CuSO4 on root initiation and elongation was also examined. Results: A significant difference was observed between control (MS medium copper level) and higher concentrations of copper sulphate incorporated into MS medium for mean number of shoots produced per explants, mean shoot length, mean leaf surface area and chlorophyll content of developing banana shoots. Maximum number of shoots per explants, highest shoot length and chlorophyll content were observed on MS medium containing 6 mg L–1 copper sulphate. Rooting of micropropagated shoots was also stimulated by copper sulphate supplement. The highest number of roots per shoot and the longest roots were noticed in rooting medium supplemented with 4 mg L–1 copper sulphate. Conclusion: These findings suggest that copper may have an important role in improving the micropropagation potential of banana.