Water shortage is the most important factor constraining agricultural production all over the world. New irrigation strategies must be established to use the limited water resources more efficiently. This study was carried out in a completely randomized design with three replications under the greenhouse condition at Shahrekord University, Shahrekord, Iran. In this study, the physiological responses of pepper plant affected by irrigation water were investigated. Irrigation treatments included control (full irrigation level, FI) and three deficit irrigation levels, 80, 60 and 40% of the plant’s water requirement called DI80, DI60, and DI40, respectively. A no plant cover treatment with three replications was also used to measure evaporation from the soil surface. Daily measurements of volumetric soil moisture (VSM) were made at each 10 cm intervals of the soil column, considered as a layer. The differences between the measured VSM and the VSM in the next day and evaporation rate at the soil surface at the same layer of the no plant cover treatment were calculated. Eventually, by considering the applied and collected water in each treatment, evapotranspiration (ETC) and root water uptake (RWU) in each layer per day were estimated. Furthermore, fruit number per plant, fresh fruit weight/day, root fresh/dry weight, shoot fresh/dry weight, root zone volume, root length and density, crop yield, and water use efficiency (WUE) were measured under different water treatments. The results showed that the maximum and minimum of all the studied parameters were found in the FI and DI40 treatments, respectively. ETC in the DI80, DI60, and DI40 treatments were reduced by 14.2, 37.4, and 52.2%, respectively. Furthermore, applying 80, 60, and 40% of the plant’s water requirement led to crop yield reduction by 29.4, 52.7, and 69.5%, respectively. The averages of root water uptakes (ARWUs) in the DI80, DI60, and DI40 treatments reduced by 17.08, 48.72, and 68.25%, respectively. WUE and crop yield also showed no significant difference in the FI and DI80 treatments. Moreover, in the DI80 treatment the reduced rate of water uptake was less than the reduced rate of plant's applied water. According to these results, it can be concluded that 20% deficit irrigation had no significant reduction on the yield of pepper, but above this threshold, there was an adverse effect on the growth and yield. Therefore, for water management in the regions with limited water resources, plant's applied water can be decreased around 20%.