The aim of this study was to investigate the physiological basis and molecular mechanism of genotypic variation in drought response of maize seedlings. Comparative physiological and proteomic analyses were conducted in the leaves of droughttolerant Liyu 35 (LY) and drought-sensitive Denghai 605 (DH) maize genotype seedlings. Drought induced a significant decrease of relative water content and osmotic potential of leaves, length and volume of roots, and total dry weight, but significantly increased malondialdehyde in DH seedlings. However, root dry weight , proline content and antioxidant enzyme activities increased more in LY than in DH. Forty-two spots in LY and 17 spots in DH that showed significant abundance variations were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. These drought-responsive proteins were mainly involved in biological processes of photosynthesis, defense and oxidative stress, carbohydrate and energy metabolism, protein synthesis and processing, and cell wall biogenesis and degradation. Among them, proteins involved in defense and oxidative stress, and protein synthesis and processing were largely enriched in the LY genotype, which may contribute to a natural variation of drought resistance between LY and DH genotypes. The altered protein abundance and corresponding physiological-biochemical response shed some light on molecular mechanisms related to drought tolerance in drought-tolerant maize and provide key candidate proteins for genetic improvement of maize.