ABSTRACT: Drought stress, especially during the seedling stage, seriously limits the growth of maize and reduces production in the northeast of China. To investigate the molecular mechanisms of drought response in maize seedlings, proteome changes were analyzed. Using an isotopic tagging relative quantitation (iTRAQ) based method, a total of 207 differentially accumulated protein species (DAPS) were identified under drought stress in maize seedlings. The DAPS were classified into ten essential groups and analyzed thoroughly, which involved in signaling, osmotic regulation, protein synthesis and turnover, reactive oxygen species (ROS) scavenging, membrane trafficking, transcription related, cell structure and cell cycle, fatty acid metabolism, carbohydrate and energy metabolism, as well as photosynthesis and photorespiration. The enhancements of ROS scavenging, osmotic regulation, protein turnover, membrane trafficking, and photosynthesis may play important roles in improving drought tolerance of maize seedlings. Besides, the inhibitions of some protein synthesis and slowdown of cell division could reduce the growth rate and avoid excessive water loss, which is possible to be the main reasons for enhancing drought avoidance of maize seedlings. The incongruence between protein and transcript levels was expectedly observed in the process of confirming iTRAQ data by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, which further indicated that the multiplex post-transcriptional regulation and post-translational modification occurred in drought-stressed maize seedlings. Finally, a hypothetical strategy was proposed that maize seedlings coped with drought stress by improving drought tolerance (via. promoting osmotic adjustment and antioxidant capacity) and enhancing drought avoidance (via. reducing water loss). Our study provides valuable insight to mechanisms underlying drought response in maize seedlings.