Meningiomas, one of the most common benign brain tumors in humans, arise from arachnoid cells in the brain meninges. Our investigations have revealed that miR-335 is a typical microRNA overexpressed in meningiomas in humans. Characterization of the effects of miR-335 overexpression in meningiomas demonstrated that elevated levels of miR-335 increased cell growth and inhibited cell cycle arrest in the G0/G1 phase in vitro; in addition, reduction of the miR-335 levels had the opposite effect on tumor growth and progression. Further, previous studies have shown that the mechanism of effect of miR-335 on the proliferation of meningioma cells is associated with alterations in the expression of human retinoblastoma 1 (Rb1). Our results indicate that miR-335 plays an essential role in the proliferation of meningioma cells by directly targeting the Rb1 signaling pathway. Thus, our results highlight a novel molecular interaction between miR-335 and Rb1, and miR-335 may represent a potential novel therapeutic agent to target the proliferation of meningioma cells.