Abscisic acid (ABA) plays a vital role in the induction of low temperature tolerance in plants. To understand the molecular basis of this phenomenon, we performed a proteomic analysis on an ABA-deficit mutant barley (Az34) and its wild type (cv Steptoe) under control conditions (25/18 °C) and after exposure to 0 °C for 24 h. Most of the differentially abundant proteins were involved in the processes of photosynthesis and metabolisms of starch, sucrose, carbon, and glutathione. The chloroplasts in Az34 leaves were more severely damaged, and the decrease in Fv/Fm was larger in Az34 plants compared with WT under low temperature. Under low temperature, Az34 plants possessed significantly higher activities of ADP-glucose pyrophosphorylase, fructokinase, monodehydroascorbate reductase, and three invertases, but lower UDP-glucose pyrophosphorylase activity than WT. In addition, concentrations of proline and soluble protein were lower, while concentration of H2O2 was higher in Az34 plants compared to WT under low temperature. Collectively, the results indicated that ABA deficiency induced modifications in starch and sucrose biosynthesis and sucrolytic pathway and overaccumulation of reactive oxygen species were the main reason for depressed low temperature tolerance in barley, which provide novel insights to the response of barley to low temperature under future climate change.