D-Ribose is active in glycation and rapidly produces advanced glycation end products, leading to cell death and to cognitive impairment in mice. Glycated serum protein (GSP) is a relatively short-term biomarker for glycemic control in diabetes mellitus. However, whether D-ribose is related to GSP is unclear. The aim of this work was to identify the contribution of D-ribose to GSP compared to D-glucose. Here, we showed that the yield of glycated human serum albumin with D-ribose was at least two-fold higher than that with D-glucose in a 2-week incubation. The glycation of human serum albumin (HSA) with D-ribose was much faster than that with D-glucose, as determined by monitoring changes in the fluorescent intensity of glycation products with time. Liquid chromatography-mass spectrometry/mass spectrometry revealed that 17 and 7 lysine residues on HSA were glycated in the presence of D-ribose and D-glucose, respectively, even when the concentration ratio [D-ribose]/[D-glucose] was 1/50. The intraperitoneal injection of D-ribose significantly increased the GSP levels in Sprague Dawley rats, but the injection of D-glucose did not. The level of D-ribose was more positively associated with GSP than the level of D-glucose in streptozotocin-treated rats. In diabetic patients, the levels of both D-ribose and D-glucose were closely related to the level of GSP. Together, these in vitro and in vivo findings indicated that D-ribose is an important contributor to the glycation of serum protein, compared to D-glucose. To assess GSP levels in diabetes mellitus, we should consider the contribution from D-ribose, which plays a nonnegligible role.