BackgroundMepiquat chloride (MC) application and plant population density (PPD) increasing are required for modern cotton production. However, their interactive effects on leaf physiology and carbohydrate metabolism remain obscure. This study aimed to examine whether and how MC and PPD affect the leaf morpho-physiological characteristics, and thus final cotton yield. PPD of three levels (D1: 2.25 plants·m−2, D2: 4.5 plants·m−2, and D3: 6.75 plants·m−2) and MC dosage of two levels (MC0: 0 g·ha−2, MC1: 82.5 g·ha−2) were combined to create six treatments. The dynamics of nonstructual carbohydrate concentration, carbon metabolism-related enzyme activity, and photosynthetic attributes in cotton leaves were examined during reproductive growth in 2019 and 2020.ResultsAmong six treatments, the high PPD of 6.75 plants·m−2 combined with MC application (MC1D3) exhibited the greatest seed cotton yield and biological yield. The sucrose, hexose, starch, and total nonstructural carbohydrate (TNC) concentrations peaked at the first flowering (FF) stage and then declined to a minimum at the first boll opening (FBO) stage. Compared with other treatments, MC1D3 improved starch and TNC concentration by 5.4% ~ 88.4%, 7.8% ~ 52.0% in 2019, and by 14.6% ~ 55.9%, 13.5% ~ 39.7% in 2020 at the FF stage, respectively. Additionally, MC1D3 produced higher transformation rates of starch and TNC from the FF to FBO stages, indicating greater carbon production and utilization efficiency. MC1D3 displayed the maximal specific leaf weight (SLW) at the FBO stage, and the highest chlorophyll a (Chl a), Chl b, and Chl a + b concentration at the mid-late growth phase in both years. The Rubisco activity with MC1D3 was 2.6% ~ 53.2% higher at the flowering and boll setting stages in both years, and 2.4% ~ 52.7% higher at the FBO stage in 2020 than those in other treatments. These results provided a explanation of higher leaf senescence-resistant ability in MC1D3.ConclusionIncreasing PPD coupled with MC application improves cotton yield by enhancing leaf carbohydrate production and utilization efficiency and delaying leaf senescence.