BACKGROUND: In this study, we evaluate whether the use of biliverdin (BV), a natural non-toxic antioxidant product of haeme catabolism, can suppress head and neck squamous cell carcinoma (HNSCC) cell proliferation and improve the tumour survival both in vitro and in vivo. Furthermore, we investigate whether this therapeutic outcome relies on BV's potent antioxidant effect on reactive oxygen species (ROS)-mediated signalling. METHODS: Two well-characterised HNSCC cell lines and a mouse model with human HNSCC were used for this study. In vitro, the effect of BV on ROS was assayed. Subsequently, critical regulatory proteins involved in growth, antiapoptotic, and angiogenic pathways were investigated by western blot analysis. In addition, the antiproliferative effect of BV was also evaluated using the clonogenic assay. Moreover, tumour growth inhibition was assessed using a mouse model with HNSCC. RESULTS: Biliverdin treatment resulted in decreased ROS, leading to suppression of proliferation and angiogenesis pathways of HNSCC, significantly decreasing the expression and phosphorylation of oncogenic factors such as epidermal growth factor receptor (EGFR), phosphorylation of Akt, and expression of angiogenic marker and transcription factor, hypoxia-inducible factor1-a (HIF1-a). Furthermore, this downregulation of ROS by BV led to a significant suppression of tumour growth in vivo. CONCLUSIONS: Our study demonstrates the efficacy of a novel therapeutic approach using BV as an antitumour agent against HNSCC through its effect on EGFR/Akt and HIF1-a/angiogenesis signal transduction pathways. Our findings indicate that BV's inhibitory effect on these tumorigenic pathways relies on its antioxidant effect, and may extend its therapeutic potential to other solid cancers.