To explore the response of mesophyll conductance (g_m) and CO₂ assimilation of wheat flag leaves to soil available phosphorus (P) deficiency in the southwest wheat production region. During the years of 2020 to 2022, the experiment was conducted in the Renshou experiment station. Straw mulching (SM) and no mulch (NSM) were the main plots. Three P levels of 0 (P0)kg/hm², 75 (P75)kg/hm² and 120 (P120) kg/hm² were set as the split plots. The responses of light response curve, mesophyll conductance and CO₂ assimilation efficiency to P levels of flag leaves of wheat were analyzed. The results show that the leaf mass per area (LMA), P content per unit area (PA), P content per unit mass (PM) and net photosynthetic rate (P_n) of flag leaf under SM were 4.1%, 16.9%, 12.2% and 6.9% respectively, greater than that in NSM, and the LMA, PA, PM and P_n of flag leaf increased with the increase of phosphorus fertilization rate. Straw mulching with phosphorus increased the maximum net photosynthetic rate (P_nmax) and apparent quantum efficiency (AQY) of flag leaves and increased the photosynthetic potential of flag leaves. Phosphorus fertilization significantly improves the actual photochemical efficiency of photosystem II (ΦPSII), the maximum carboxylation efficiency (V_cmax), ribulose 1,5-diphosphate carboxylase (Rubisco), intercellular CO₂ concentration (C_i), chloroplast CO₂ concentration (C_c), stomatal conductance (g_s) and g_m of flag leaf. These value under P75 and P120 were 16.8%-23.8%, 27.9%-35.3%, 30.9%-61.7%, 1.4%-3.6%, 4.8%-13.1%, 9.0%-10.2% and 16.0%-16.9% higher than that under P0, respectively. There was no significant difference between P75 and P120. Straw mulching and phosphorus fertilization decreased stomatal limitation (L_s) and mesophyll limitation (L_m), and biochemical limitation (L_b) was the main limiting factor of photosynthetic rate in flag leaves. In conclusion, straw mulching combined with 75 kg/hm² can improve the phosphorus content, resulting in the enhancement of the actual photochemical efficiency of photosystem II, mesophyll conductance and CO₂ assimilation efficiency of flag leaves of southwest winter wheat, reduce stomatal restriction and mesophyll restriction, and thus increase the net photosynthetic rate of flag leaves.