Resistance to chemotherapy is a major obstacle in clinical oncology. Hypoxia is a hallmark of solid tumours as a result of poorly structured tumour neovasculature and is known to be a key contributor to cancer malignancy and reduced drug effectiveness. Hypoxia-inducible factor-1 (HIF-1) mediates the hypoxic response, bringing about adaptive responses to the change in microenvironment. This study investigated the effect of 24 h hypoxia on the sensitivity of SH-SY5Y neuroblastoma cells to 5-fluorouracil (5-FU). Cytotoxicity, apoptosis and membrane integrity cell-based assays were carried out to observe changes in cell viability alongside apoptotic and necrotic cell death to determine the molecular mechanisms underlying reduced drug sensitivity. Hypoxic cells showed no change in cell viability to 5-FU in comparison to normoxic controls, suggesting that hypoxia conferred reduced drug sensitivity. As a measure of apoptosis, caspase-3/7 levels were significantly higher in hypoxic cells treated with increasing 5-FU concentrations. However, this was associated with less cell necrosis, suggesting that despite increased hypoxia-induced apoptosis, a significant decrease in uncontrolled cell death accounted for the change in viability observed. This finding also implies that alternative cell survival mechanisms could also be activated.