Cancer stem cells (CSCs) have been identified to exert tumor-initiating ability, resulting in the recurrence, metastasis and chemoresistance of oral squamous cell carcinomas. In the present study, we showed that GMI, an immunomodulatory protein from Ganoderma microsporum, induc ed a cytotoxic effect in oral carcinomas stem cells (OCSCs). Treatment of GMI dose-dependently inhibited the expression of CSC markers, including ALDH1 activity and CD44 positivity. Moreover, GMI suppressed the self-renewal property, colony formation, migration, and invasion abilities as well as potentiated chemo-sensitivity in OCSCs. Our results suggested that the tumor suppressive effect of GMI was mediated through inhibition of IL-6/Stat3 signaling pathway. Furthermore, tumor growth was reduced in mice bearing xenograft tumors after oral administration of GMI. Taken together, we demonstrated the anti-CSC effect of GMI in oral cancer and GMI may serve as a natural cisplatin adjuvant to prevent cancer recurrence.