Further research related to the role of plant-associated bacteria at the molecular level, gene regulation, modulation, and function can lead to hope for enhancing the growth and performance of agricultural plants in harsh environment. Two studies were performed to investigate the function of Azospirillum brasilense in the regulation of proline-responsive gene expression, as well as some important physiological and biochemical traits related to drought resistance in wheat. Wheat seedlings grown from inoculated and uninoculated seeds were grown under no-water-limitation condition for 21 days and then were subjected to two water regimes: 80% of soil water-holding capacity (WHC) and 25% of WHC. The relationship between A. brasilense inoculation and proline accumulation caused by delta-1-pyrroline-5-carboxylate synthase (P5CS) as well as antioxidant system defense was elucidated 2 weeks after water-deficit imposition. Experiment 2 was conducted under greenhouse condition to assess the modulation of photosynthetic traits, cell membrane stability, and relative water content of leaves as well as grain yield in repose to A. brasilense inoculation. Treatments performed in the two experiments were the same, except water-deficit condition in experiment 2 was imposed at anthesis stage. Under water-deficit conditions, inoculation led to lower accumulation rate (82%) of P5CS mRNA as compared to control plants. Under water-deficit condition, inoculated plants showed lower (13.8%) content of proline compared to control plants. Inoculation alleviated the negative effect of water deficiency by increasing the antioxidant enzyme activity as it increased the activity of peroxidase by 11.7%, glutathione peroxidase by 7.3%, catalase by 65% and glutathione reductase by 70% as compared to uninoculated plants. Under water deficiency, plants inoculated with A. brasilense maintained higher photosynthetic parameters such as net carbon dioxide assimilation rate (84%), stomatal conductance (182%), and sub-stomatal carbon dioxide concentration (69%) compared to uninoculated plants. The present study confirmed that expression of the evaluated gene (P5CS) along with the accumulation of proline was a common response of wheat to water deficiency. Both experiments conducted confirmed that. The inoculated plant displayed a lower level of the evaluated gene and proline content, which can be considered for future research and could open a new hope for improving crop resistance to water-deficit stress.