High temperature stress (HT) significantly reduces maize yield by impairing starch accumulation in kernels. However, the mechanism by which HT affects starch synthesis remains controversial-whether through reduced assimilate supply or direct inhibition on kernel metabolism. To clarify these mechanisms, a heat-sensitive maize hybrid, Xianyu 335 (XY), was exposed to 30°C/20°C (maximum/minimum temperature, control) and 40°C/30°C for seven consecutive days during the seed setting stage. Synchronous pollination (SP), apical pollination (AP), and shading treatments were applied to manipulate the inherent source–sink ratio in maize plants. Results showed that apical kernel weight decreased by 11.9% under 40°C in the SP treatment. The ¹³C content, starch accumulation, and cell-wall invertase (CWIN) activity also declined by 15.9, 36.7, and 16.4%, respectively, under HT. Inthe shading treatment, 40°C/30°C caused even greater reductions in ¹³C content, starch accumulation, and CWIN activity due to diminished assimilate supply. Conversely, inthe AP treatment, starch content and CWIN activity increasedby 22.0 and 18.5%, respectively, under 40°C/30°C, resulting in kernel weight and ¹³C content similar to those in SP and shading treatments regardless of temperature. Consistent with apical kernels under AP, HT did not negatively affect middle kernels in either SP or shading treatments, as kernel weight and starch content remained unchanged under HT. Although all kernels were exposed to the same HT or controlenvironment, their responses varied a lot. The impaired starch synthesis in apical kernels under HT was rescued by increasing carbon supply viaAP treatment. The contrasting performance among middle kernels, apical kernels under AP, and apical kernels under SP or shading indicates that reduced carbon supply is a critical factor underlying inhibited starch accumulation. Our findings provide a theoretical basis for further understanding kernel abortion under HT.

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