Prohexadione-calcium (Pro-Ca) has been shown to positively regulate crop tolerance to saline–alkali stress. However, the optimal concentration of Pro-Ca application and the mechanisms through which it enhances saline–alkali tolerance and yield in soybean remain unclear. This study aimed to determine the optimal concentration of exogenously applied Pro-Ca and reveal the mechanisms underlying Pro-Ca’s effect on remediation and yield response in soybean under saline–alkali stress. The results indicated that saline–alkali stress negatively impacted the morphological and physiological traits of soybean seedlings by triggering the production of reactive oxygen species (ROS), leading to oxidative damage of the grana lamellae due to excessive accumulation of Na⁺. An application of 100 mg L⁻¹ Pro-Ca was found to be optimal, promoting dry matter accumulation and normalized difference vegetation index (NDVI) by significantly reducing Na⁺ uptake under saline–alkali stress. Moreover, integrated physiological, ultrastructural, and transcriptomic analyses indicated that Pro-Ca significantly enhanced the ascorbate–glutathione (AsA–GSH) cycle by up-regulating the expression of related genes to enhance the activities of ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and the AsA/dehydroascorbate (DHA) and GSH/oxidized glutathione (GSSG) ratios to quench ROS, thereby protecting both thylakoid and mitochondrial membranes from degradation. The differentially expressed genes (DEGs) encoding ascorbate and aldarate metabolism were significantly (P<0.05) enriched in the integral component of the membranes. Furthermore, Pro-Ca treatment up-regulated the expression of genes encoding photosystems under saline–alkali stress, thereby reducing the photoinhibition and stomatal limitation (L_s), mitigating damage to photosystems, and preventing yield reduction. In summary, foliar application of Pro-Ca could efficiently enhance soybean seedlings’ tolerance to saline–alkali stress by inhibiting Na⁺ influx, enhancing the AsA–GSH cycle, maintaining the biomembrane system, and improving photosynthetic efficiency.

soybean  /  prohexadione-calcium  /  zsaline–alkali stress  /  physiological  /  ultrastructural  /  transcriptomic