Summary During recent years, the opportunity to use energy crops for the production of biofuels aroused many interest by virtue of the potential reduction of green house gasses emissions associated to their large-scale use. Nevertheless, many studies highlight that the substitution of fossil fuels with biofuels from energy crops can lead up to serious problems related to the pressure on water resources. The present research had the aim to investigate the relations existing between biofuels production and the pressure on water resources, and how these relations are affected by climate variability. To this aim, the water footprint of maize cultivation in Tuscany (central Italy) was computed and its trend was analyzed during the last 55 years in relation to climate variability with particular attention to precipitations. The results demonstrated that the WF was affected by climate variability mainly through the effect of climate on the crop cycle. The total WF decreased over time but an increase of the blue component was found due to the change in precipitation patterns and to the rise of temperatures. Concerning the gray WF, despite a decrease in the last 55-years period mainly due to precipitation decrease, there was a reduction in N uptake by the crop and an accumulation of nitrate into the soil.