Inoculation with arbuscular mycorrhizal (AM) fungi can modify stomatal behavior and increase antioxidant enzyme activities and therefore play a pivotal role in plant growth. We hypothesised that inoculation with AM fungi postpones the non-hydraulic root signal and alters physiological and biochemical traits, which enhances drought tolerance and water-use efficiency (WUEB) for plant biomass. Two pot experiments (including progressive soil drying and partial root-zone drying) were conducted to reveal how mycorrhizal colonization altered root signal and its effects on plant growth, biochemical traits and WUEB in maize seedlings in drying soil. In our experiments, inoculation with Funneliformis mosseae improved water absorption and reduced the sensitivity of roots to drought. In addition, it decreased leaf abscisic acid (ABA) content of inoculated plants. Regardless of water conditions, plant biomass production, antioxidant enzyme activity, net photosynthetic rate, stomatal conductance and WUEB were elevated in AM fungal treatments compared to non-AM fungal treatments. Under water-stressed conditions, inoculation with F. mosseae greatly reduced leaf ABA content, and postponed the decline in photosynthetic rate, stomatal conductance and osmotic adjustment. Malondialdehyde (MDA) level was significantly lower in mycorrhizal plants than in non-inoculation plants. However, inoculation with F. mosseae increased antioxidant enzyme activities including peroxidase (POD) and superoxide dismutase (SOD). In this study, inoculation with F. mosseae reduced ABA accumulation that acts as a non-hydraulic root signal and thereby postponed a decline in stomatal conductance and photosynthetic rate, improved water use efficiency and antioxidant enzymes activities, and accordingly reduced proline and MDA content. Thus, inoculation with AM fungi played a role in effective defense for better drought acclimation in water-stressed maize seedlings.