Phosphorus is one of the three essential nutrients for crops. It should also be noted that rock phosphate is a nonrenewable resource that is being depleted. More understanding is needed about how P affects wheat yield and quality. Pores and channels in starch granules play an important role in starch biosynthesis and physicochemical characteristics ofwheat. This study was conducted to reveal the changes of micro-structural characteristics of starch granules and the mechanisms under different phosphorus application rates in wheat. In this study, the winter wheat cultivar ‘Xindong 20’ were used in this study. On160 d after sowing (about 5% plants returning green), three levels of phosphorus treatments, i.e., 0 kg P2O5/ha;105 P2O5/ha; and 210 P2O5/ha, were applied, and the samples were collected on the 7, 14, 21, 28 and 35 days postanthesis. As the results, scanning electron microscopy showed that P fertilizer amount (0, 105, and 210 kg P2O5/ha)had no significant effect on the shape of starch granules in wheat grain. However, confocal laser scanning microscopy indicated that P amount influenced the microstructure of the starch granules. In response to 105 P2O5/ha treatment, the “pinholes” on the granules were easier to find, and the fluorescence from the starch granules was stronger and clearly visible. At 6 days post germination, the number of “pinholes” and pits on the starch granules in 105 P2O5/ha treatment was most abundant. After exogenous amyloglucosidase digestion, the A-type starch granules developed under 105 P2O5/ha or 210 P2O5/ha conditions were more prone to be broken into halves than those formed under 0 P2O5/ha conditions. Starch granules from the 105 kg P2O5/ha treatment released significantly more reducing sugars than those from the 0 and 210 kg P2O5/ha treatments during digestion with alpha-amylase and amyloglucosidase digestion. Phosphorus application (i.e. the

105 and 210 kg P2O5/ha treatments) significantly increased the relative expression of genes related to starch synthesis (especially during early to mid-grain filling) and starch degradation (especially during mid- and late grain filling). Phosphorus application also increased the transcript abundance of amylase genes at the periphery of the endosperm. We propose that P application, especially the 105kg P2O5/ha treatment, enhanced channels in wheat starch granules. These channels facilitated the transport of substances required for starch biosynthesis, thus increasing starch accumulation in wheat endosperm. These results provide insight into the potential mechanisms through which P influences the microstructure and biosynthesis of wheat starch.