Butyric acid produced in the intestine by butyric acid-producing bacteria (BAPB) is known to suppress excessive inflammatory response and may prevent chronic disease development. We evaluated whether fiber-rich barley intake increases BAPB in the gut and concomitantly butyric acid in feces. Eighteen healthy adults received granola containing functional barley (BARLEYmax®) once daily for four weeks. Fecal DNA before intake, after intake, and one month after intake was analyzed using 16S rRNA gene sequencing to assess microbial diversity, microbial composition at the order level, and the proportion of BAPB. Fecal butyric acid concentration was also measured. There were no significant differences in diversities and microbial composition between samples. The proportion of BAPB increased significantly after the intake (from 5.9% to 8.2%). However, one month after stopping the intake, the proportion of BAPB returned to the original value (5.4%). Fecal butyric acid concentration increased significantly from 0.99 mg/g feces before intake to 1.43 mg/g after intake (p = 0.028), which decreased significantly to 0.87 mg/g after stopping intake (p = 0.008). As BAPB produce butyric acid by degrading dietary fiber, functional barley may act as a prebiotic, increasing BAPB and consequently butyric acid in the intestine.