Overexpression of survivin plays a crucial role in tumorigenesis and correlates with poor prognosis in human malignancies. Thus, survivin has been proposed as an attractive target for new anti-tumor interventions. A natural product library was used for natural compound screening through MTS assay. The expression of survivin in oral squamous cell carcinoma (OSCC) and the inhibitory effect of xanthohumol (XN) on OSCC were examined by anchorage-dependent and -independent growth assays, immunoblot, immunofluorescence, immunohistochemical staining, ubiquitination analysis, co-immunoprecipitation assay, CRISPR-Cas9-based gene knockout, and xenograft experiment. Survivin is highly expressed in OSCC patient-derived tissues and cell lines. Knockout of survivin reduced the tumorigenic properties of OSCC cells in vitro and in vivo. With a natural compound screening, we identified that xanthohumol inhibited OSCC cells by reducing survivin protein level and activating mitochondrial apoptotic signaling. Xanthohumol inhibited the Akt-Wee1-CDK1 signaling, which in turn decreased survivin phosphorylation on Thr34, and facilitated E3 ligase Fbxl7-mediated survivin ubiquitination and degradation. Xanthohumol alone or in combination with radiation overcame radioresistance in OSCC xenograft tumors. Our findings indicate that targeting survivin for degradation might a promising strategy for OSCC treatment.