.Objectives: To measure phosphorus metabolites in human parotid glands by 31P-MRS using three-dimensional chemical-shift imaging (3D-CSI), and ascertain whether this method can capture changes in adenosine triphosphate (ATP) and phosphocreatine (PCr) levels due to saliva secretion. Study Design: The parotid glands of 20 volunteers were assessed by 31P-MRS using 3D-CSI on 3T MRI. After obtaining a first (baseline) measurement, the participants took vitamin-C tablets and measurements were obtained twice more, in a continuous manner. The peak area ratios of PCr and β-ATP were evaluated. Results: A high proportion of PCr (0.26 ± 0.08) and ATP (α: 0.16 ± 0.06; β: 0.27 ± 0.06; γ: 0.21 ± 0.03) was noted at baseline. A significant decrease in β-ATP was observed between baseline (“pre”; 0.27 ± 0.06) and the first scan after vitamin-C stimulation (“post-1”; 0.19 ± 0.07, p Conclusions: 31P-MRS with 3D-CSI can assess the major phosphorus metabolites in human parotid glands and capture changes in their levels due to saliva secretion. This technique is simple, non-invasive, and provides new information regarding saliva secretion.