The aim of our work was to use a targeted GC-MS approach to investigate the difference in organic acid profiles between individuals with impaired fasting glucose (IFG) and healthy controls and then to investigate the alterations in postprandial organic acid profiles after a meal of highland barley (HB) in IFG individuals. Firstly, 30 IFG and 30 healthy individuals were recruited and 26 organic acids were detected to characterize the organic acid profiles in the fasting serum metabolome. Secondly, 15 participants of the IFG group received three different loads: glucose (GL), white rice (WR) and HB. Serum was collected at time zero, 30, 60, 90 and 120 min after the test load. The results showed fasting organic acid profiles were different between the IFG group and the controls. For the postprandial changes in organic acids after the three test loads, six organic acids related to the tricarboxylic acid (TCA) cycle, namely citrate, cis-aconitic acid, fumarate, succinate, pyruvate and malate, had a significant test load effect (p < 0.01) and a significant time × test load interaction effect (p < 0.01). The AUC0-120 min values for citrate, fumarate and malate after WR and HB loads were significantly lower (p < 0.05) compared to the GL load. In addition, the AUC0-120 min value for pyroglutamic acid after WR and HB loads was significantly higher (p < 0.05) compared to the GL load, whereas the AUC0-120 min for malonic acid after WR and HB loads was significantly lower (p < 0.05) compared to the GL load. Altogether, these findings suggest that the HB load producing low postprandial glucose and insulin responses brings about several alterations in organic acids.