Pterostilbene, a polyphenolic compound and an analog of resveratrol, exerts various biological activities and has higher bioavailability and metabolic stability than resveratrol. However, the effectiveness of pterostilbene intake in humans, particularly its effect on blood microRNA (miRNA) expression levels, has not been evaluated. Accordingly, this pilot study aimed to investigate the effects of pterostilbene on blood biochemistry and blood miRNA expression levels and the safety of continuous intake at doses of 10 or 100 mg/d over 12 wk. A double-blind, placebo-controlled parallel-arm comparison trial was conducted with 30 healthy men. In the analysis of blood miRNA expression levels, miR-34a and miR-193b showed very high increases at week 4 and after week 4 of intake, respectively, suggesting that the responders might be present among participants in the pterostilbene intake group. No adverse events were reported during the trial in any participant, and no abnormalities were observed upon examination by the responsible physician. Thus, pterostilbene intake would regulate blood miRNA expression levels, and the results can be utilized in human studies investigating miRNA expression levels with functional food ingredients.