MicroRNAs (miRNAs) have been implicated to play important roles in the regulation of the malignant progression of cancer. In this study, we examined whether and how the miR-520b family participates in the malignancy of head and neck cancer (HNC). We found that miR-373/520a/520c were up-regulated, while miR-302b/520b/520h were down-regulated in HNC cell lines. Mir-520b was then investigated further. The transduction of HNC cells with exogenous miR-520b significantly increased their sensitivity to therapeutic drugs and radiation treatment. The transduction of cells with miR-520b also decreased cell motility, which was accompanied by the reversion of the epithelial-mesenchymal transition. Significantly, miR-520b suppressed the conversion of cancer stemness as demonstrated by the suppression of spheroid cell formation, the reduction in the expression of stemness markers (nestin and CD44), and the inhibition of pluripotent regulators (Twist, Nanog and Oct4). Furthermore, the CD44 protein was identified as the direct target of miR-520b, which was shown by the response to miR-520b modulation via transductions with exogenous miR-520b or with antagomirs. Functional analyses also confirmed this regulatory axis. The altered cell motility and spheroid formation induced by miR-520b were rescued upon CD44 suppression. The stemness regulators induced by miR-520b were also inhibited when CD44 was silenced. An in vivo study in mice showed that miR-520b knockdown led to an acceleration of tumorigenesis and liver colonization. Conversely, miR-520b transduction dramatically restrained tumor growth and decreased metastasis. Taken together, our study demonstrated that miR-520b inhibits the malignancy of HNC through the regulation of cancer stemness by targeting CD44. MiR-520b may therefore serve as an emerging therapeutic target that may be further developed for the intervention of refractory HNC.