The quality and quantity of the water used in agriculture are important factors to ensure sustainable agricultural production. It is crucial to determine the responses of plants under deficit irrigation conditions in the arid and semi-arid areas to varying salinity of irrigation water. The responses of spinach were investigated in terms of agronomic and physiological aspects to different water salinities and water stress levels. In this study, a total of 15 experimental treatments were carried out, including five different levels of water salinity (S1, 0.3 dS/m; S2, 1.5 dS/m; S3, 3.0 dS/m; S4, 4.5 dS/m and S5, 6.0 dS/m) and three different irrigation levels (I100; full irrigation, I75; 25% water deficit, and I50; 50% water deficit). The salinity of irrigation water had statistically significant effects on the fresh and dry weight, leaf area, membrane damage, color pigments, and carotenoid values in spinach. Similarly, irrigation water salinity has a significant effect on enzyme activities such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and malondialdehyde (MDA), as well as proteins, proline, and hydrogen peroxide (H2O2) contents. Furthermore, the application of different irrigation levels in spinach; resulted in a significant difference in the fresh and dry weights, the number of leaves per plant, leaf area, leaf water content, and color pigments. Additionally, enzyme activities such as CAT, SOD, and MDA, as well as protein and proline content, were significantly affected by different irrigation levels. The highest yield response factor (ky) calculated in different irrigation water salinity applications was 1.79 in the S1 application, followed by 1.32 in the S2 (1.5 dS/m) application. It was found that ky values in spinach were less than 1.00 in irrigation with an EC of 3.0 dS/m and above; thus, making it tolerant to water shortages. In the case of irrigation with saline water such as 4.5 and 6.0 dS/m, the yield at I75 (25% water stress) irrigation level was found closely related to the treatments of full irrigation or even slightly higher. Therefore, in the water shortage regions with saline irrigation water, 25% water restriction on spinach has been proposed as an important strategy for the protection of water resources as well as control of soil salinity.