Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide. Let-7 family has been shown to function as a tumor suppressor through regulating multiple oncogenic signaling. Recent study reported that combined underexpression of miR-205 and let-7d showed negative correlation with the survival prognosis of head and neck cancer patients. However, the let-7d-involved mechanism in regulating OSCC is still unclear. In this study, we first demonstrated that let-7d expression was significantly decreased while Twist and Snail expression was increased in OSCC cancer cell lines and primary cultures as compared to normal human oral keratinocyte cells. To further investigate the role of let-7d in OSCC, we applied the SPONGE method to knock down let-7d in OECM-1 and two primary OSCC cell types. The results showed that knockdown of let-7d promote epithelial-mesenchymal transition (EMT) traits and migratory/invasive capabilities in OSCC cells. Furthermore, down-expression of let-7d significantly activated Twist and Snail expressions and chemo-resistant abilities of OSCC cells. Notably, overexpression of let-7d effectively reversed the EMT phenotype, blocked migratory/invasive abilities, and further increased the chemosensitivity in oral cancer tumor initiating ALDH1+ cells. In sum, these results show that let-7d negatively modulates EMT expression and also plays a role in regulating chemo-resistant ability in oral cancer.