Cytoglobin has been implicated in a range of pathological conditions including fibrosis and cancer, where cytoglobin expression is linked to hypoxia- and drug-resistance. One disease where there is evidence of a role for cytoglobin is head and neck squamous cell carcinoma, a disease with a poor prognosis and where clinical resistance to platinum-based drugs is common. In the current study, we demonstrate that 3D-culture of head and neck cancer cells (PE/CA-PJ41, Liv-22K) and normal oral keratinocyte cells (NOK-hTERT) results in changes in sensitivity to cisplatin cytotoxicity compared to 2D-cultures. Relative to 2D-cultures, 3D-cultures of PE/CA-PJ41 and NOK-hTERT cells demonstrated increased cisplatin resistance. In contrast, 3D-cultures of Liv-22K cells were more sensitive to cisplatin. Evidence for cisplatin genotoxicity was observed in the form of GADD45A activation in PE/CA-PJ41 cells but not the other two cell types investigated. Furthermore, DNA-strand breaks were also detected by the alkaline comet assay in PE/CA-PJ41 cells although levels were not significantly affected by 3D-culture. Cytoglobin expression levels were elevated by 3D-culture in all three cell lines investigated and there were also changes in expression of genes related to cell division (MKI57, GJB6), cell adhesion (CDH1), stress response (NFkB, NQO1) and apoptosis (Casp3) but these changes were cell line specific. We also observed consistent transcriptional activation of HIF1a in 3D-cultures of all three cell lines, suggestive of hypoxic conditions in spheroids. In support of a direct role of hypoxia in cytoglobin induction, the HIF1a stabiliser cobalt chloride also induced cytoglobin expression in spheroids. Transcriptomic profiling of PE/CA-PJ41 cells over-expressing cytoglobin identified 121 differentially regulated genes, when cells were cultured under hypoxic conditions. Major changes identified included, upregulation of G1/S cell cycle regulation as well as wnt- and RhoGTPase-signalling pathways. In conclusion, 3D-cultures are a useful model to further study the biological function of cytoglobin in head and neck cancer and we provide evidence that further supports a role for cytoglobin in hypoxia-dependent cellular proliferation and phenotypic changes that could contribute to cisplatin resistance in vivo.