Increased climate variability is affecting crop yield and quality. Ameliorating this predicament will require an investigation of how plants respond to stresses such as a reduced supply of water. Using 10-day-old wheat seedlings, we withheld irrigation for 3 to 9 d. Their performance was then compared with untreated control plants (standard watering schedule). At each sample point, proteins were extracted from the chloroplasts, separated by two-dimensional electrophoresis (2-DE), and stained with Coomassie brilliant blue. During short-term water stress, rates of photosynthesis and transpiration, stomatal conductance, and relative water content decreased while the proline content increased. Through 2-DE, we detected 20 differentially expressed proteins from the chloroplasts and analyzed them with high through-put MALDI-TOF/TOF-MS. Under these stress conditions, 9 proteins were up-regulated in their protein abundance while the levels of 11 proteins were unevenly affected. Levels of the Rubisco small and large subunits, plasma membrane proteins in the chloride carrier/channel family, and H+-transporting two-sector ATPase were up-regulated by this treatment whereas other plasma membrane proteins, e.g., membrane-bound ATP synthase subunit b, were down-regulated by drought. Single-pass transmembrane domain such as the cytochrome b6-f complex declined in abundance under water stress. These results suggest that members of the chloride carrier/channel family as well as H+-transporting two-sector ATPase are target proteins that are induced by water stress in wheat chloroplasts.