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Abstract: The aim of this study was to estimate the increase in industrial water consumption and withdrawal in a conventional sugarcane ethanol mill due to the introduction of second-generation ethanol production by a bagasse hydrolysis process, and to identify opportunities of water reuse, in order to minimize water withdrawal. Simulations in ASPEN PLUS® software were performed for mass and energy balances. Three cases were evaluated: a conventional ethanol production plant (Case I), and two second-generation plants incorporating bagasse hydrolysis differing only in their glucose concentration processes, namely by evaporation (Case II), and by membrane separation (Case III). Results show that external withdrawals of 738L/t of cane for Case I, 955L/t of cane for Case II and 853L/t of cane for Case III are required to cover the water deficit of the plant. These values are lower than the mandated limit of 1000L/t of cane for the sugar cane industry in the State of São Paulo. Moreover, for Cases II and III, which need large additional amounts of water for the hydrolysis stage, water usages of 10.77 and 9.38L of water per litre of ethanol produced were achieved, approaching the figure of 9.34L water per litre of ethanol produced by the conventional plants (Case I). This highlights the high potential for reduction practices based on the concept of energy and water integration. [Copyright &y& Elsevier]