Phosphorylation of glucose is the prime step in sugar metabolism and energy storage. Two key glucose phosphates are involved, that is, glucose 6-phosphate (G6P) and alpha-glucose 1-phosphate (alpha G1P). The chiral conformation of glucose, G6P, and alpha G1P plays an essential role in enzyme-mediated conversions. However, few techniques were able to give a direct view of the conformational changes from glucose to G6P and alpha G1P. Here, Raman optical activity (ROA) was used to elucidate the stereochemical evolution of glucose upon phosphorylation. ROA was found to be extremely sensitive to different phosphorylation sites. A characteristic ROA marker of (+)980 cm(-1) originated from the phosphate group symmetric stretching vibration, is observed for alpha G1P with phosphorylation at chiral C1, while Phosphorylation-induced gauch-gauch (gg)/gauch-trans (gt) rotamer distribution changes no corresponding ROA signal for G6P with phosphorylation at achiral C6 is observed. can be sensitively probed by the sign of the ROA band around 1460 cm(-1). A positive ROA band at 1465 cm(-1) of glucose corresponds to a higher gt ratio, while a negative band at 1455 cm(-1) of G6P suggests a dominant gg population, and the disappearance of this ROA band for alpha G1P indicates a nearly balanced gg/gt distribution. Meanwhile, the phosphorylation at C6 and C1 could cause dramatic reduction of the conformational flexibility of the adjacent C4-OH and C2-OH, respectively. These stereochemical changes revealed by ROA spectra offer a structural basis on the understanding of sugar phosphorylation from the perspective of chirality.