Using functional near-infrared spectroscopy (fNIRS), we investigated regional brain activity corresponding to the perception of sweetness. Six subjects without taste deficit participated in the study. Subjects ate a piece of sweet snack with optical probes attached on their bilateral prefrontal area for two times, between which their mouth was rinsed with gymnemic acid (GA) solution to inhibit the sweet taste receptors on the tongue. GA rinse significantly suppressed the perceived intensity of sweetness. Statistical parametric mapping of oxy-hemoglobin signals indicated that eating sweet food activated the bilateral primary taste area (Brodmann Area (BA) 43), inferior frontal gyrus (BA44, 45), and dorsolateral prefrontal cortex (BA46), and the left premotor area (BA6). Inhibition of sweetness suppressed all of these activities except that in the right dorsolateral prefrontal cortex. These results suggest that the activation of the primary taste area and the inferior frontal gyrus plays a significant role in the perception of sweetness. The remained activity in the dorsolateral prefrontal cortex is likely to reflect the taste-related working memory process of the task in which subjects memorized and compared the sweetness of the food stimuli before and after the GA rinse. Our experiment also showed that fNIRS is feasible to investigate the neuronal activity during eating food, a situation which is very close to our real-world behavior.