In order to get a better understanding of protein functions related to water uptake in maize, we investigated the proteome dynamics of cv. Anyu308 root system at three key zones divided into Upper root (0cm-20cm), Middle root (20cm-40cm), and Lower root (40cm-60cm) by label-free quantitative shotgun proteomic approach (LFQ). A total of 489 differentially abundant proteins (DAPs) were identified in cv. Anyu308 root in a pairwise comparison of MR, LR, and UR, including shared and unique DAPs in the three root zones, respectively. Cluster analysis revealed MR and LR had similar protein expression patterns different from UR. Comparisons of protein profile indicate that more protein abundance changes occurred in MR, whereas more DAPs were downregulated in the LR and UR. Expression patterns, functional category, and pathway enrichment analyses highlight chromatin structure and dynamics, ribosomal structures, polysaccharide metabolism, energy metabolism, transport, inorganic ion transport, intracellular trafficking, and vesicular transport posttranslational modification as primary biological processes related to maize root water uptake. Furthermore, the accumulation of histones, ribosomal proteins, and aquaporins, including mitochondrion electron transport proteins and the TCA cycle, underpinned MR's high-water uptake. The results also indicated that the proteins involved in vascular transport, protein folding, and proteolysis supported the radial transport of solute across cell membranes in MR. Parallel reaction monitoring (PRM) analysis confirmed the expression profile of the proteins obtained by LQF-based proteomics. The current results provided an insight into the mechanisms of enhancing root water uptake in maize.