Background Growing evidence indicates that prolonged sedentary behaviour increases the risk of several chronic health conditions and all-cause mortality. Sedentary behaviour is prevalent among adults in the UK. Quantifying the costs associated with sedentary behaviour is an important step in the development of public health policy. Methods National Health Service (NHS) costs associated with prolonged sedentary behaviour (≥6 hours/day) were estimated over a 1-year period in 2016-2017 costs. We calculated a population attributable fraction (PAF) for five health outcomes (type 2 diabetes, cardiovascular disease [CVD], colon cancer, endometrial cancer and lung cancer). Adjustments were made for potential double-counting due to comorbidities. We also calculated the avoidable deaths due to prolonged sedentary behaviour using the PAF for all-cause mortality. Results The total NHS costs attributable to prolonged sedentary behaviour in the UK in 2016-2017 were £0.8 billion, which included expenditure on CVD (£424 million), type 2 diabetes (£281 million), colon cancer (£30 million), lung cancer (£19 million) and endometrial cancer (£7 million). After adjustment for potential double-counting, the estimated total was £0.7 billion. If prolonged sedentary behaviour was eliminated, 69 276 UK deaths might have been avoided in 2016. Conclusions In this conservative estimate of direct healthcare costs, prolonged sedentary behaviour causes a considerable burden to the NHS in the UK. This estimate may be used by decision makers when prioritising healthcare resources and investing in preventative public health programmes.