In many areas of the world, water stress is the major constraint limiting the productivity of sugarcane. The objective of this study was to identify proteins that were differentially expressed in sugarcane leaves in response to a water deficit treatment to describe the sugarcane responses at the cellular and molecular levels. Drought-tolerant sugarcane cultivar Khon Kaen 3 stalk cuttings were grown under a controlled environment in a growth chamber where a water deficit treatment was imposed by withholding watering for 5 days. The treatment group continuously received an adequate water supply. Soil moisture content (SMC), leaf water potential (LWP) and relative water content (RWC) were recorded to quantify the water deficit stress. Leaf proteins from non- and water-stressed plants were separated using two-dimensional electrophoresis (2-DE). Image analysis was performed on the electrophoresis gel to locate proteins that were differentially expressed between treatments, which were then identified using liquid chromatography coupled with electrospray ionization ion trap subjected to mass spectrometry/mass spectrometry analysis (LC-ESI-IT-MS/MS) and characterized. Two proteins involved in light-dependent reactions, chlorophyll a-b binding protein 1B-21 and chloroplastic and oxygen-evolving enhancer protein 1, were up-regulated by the stress treatment. Enzymes known to participate in antioxidant networks, including chloroplastic copper-zinc superoxide dismutase (CuZn-SOD), two-cysteine peroxiredoxin (2-Cys Prx) BAS1, superoxide dismutase [manganese] (SOD [Mn]) 3.1, SOD [Mn] 3.4, and isoflavone reductase (IFR) homolog IRL, were also up-regulated. These enzymes all used NADPH as the reducing equivalent, suggesting that linear electron flow (LEF) may dominate the total electron transport activities in sugarcane leaves under water deficit. In addition, two isoforms of ATP synthase beta and ATP synthase alpha subunits were up-regulated under water deficit, indicating that LEF was coupled to the generation of electrochemical gradients across the thylakoid membranes, leading to an increased abundance of ATP synthase beta and ATP synthase alpha subunits. There was increased abundance of a 16.9 kDa class I heat shock protein (HSP) and two isoforms of the elongation factor (EF-Tu) proteins, which are associated with heat tolerance. The identification of proteins regulated by water stress could lead to a better understanding of the cellular response to dehydration, which is an important and fundamental part of improving the stress tolerance of crops.