Background The mechanisms by which mandibular advancement splints (MAS) improve obstructive sleep apnoea (OSA) are not well understood. This study aimed to evaluate the mechanism of action of MAS by assessing their effect on upper airway structure in patients with OSA. Methods Patients were recruited from a sleep disorders clinic for treatment with a custom-made MAS. MRI of the upper airway was performed during wakefulness in the supine position, with and without the MAS. Results Sixty-nine patients with OSA were recruited. Treatment with the MAS reduced the apnoea–hypopnoea index (AHI) from 27.0±14.7 events/h to 12.2±12.5 events/h (p<0.001). There was an increase in the total airway volume with mandibular advancement (16.5±0.7 cm³ vs 18.1±0.8 cm³; p<0.01) that occurred predominantly because of an increase in the volume of the velopharynx (5.7±0.3 cm³ vs 6.5±0.3 cm³; p<0.001). This increase in airway calibre was associated with an increase in the lower anterior facial height (6.8±0.1 cm vs 7.5±0.1 cm; p<0.001), reduction in the distance between the hyoid and posterior nasal spine (7.4±0.1 cm vs 7.2±0.1 cm; p<0.001), lateral displacement of the parapharyngeal fat pads away from the airway (right parapharyngeal fat pad 0.17±0.02 cm; left parapharyngeal fat pad 0.22±0.02 cm) and anterior movement of the tongue base muscles (0.33±0.03 cm). Subanalyses in responders and non-responders to MAS treatment showed that the increase in upper airway calibre with mandibular advancement occurred only in responders. Conclusion These results suggest that the mechanism of action of MAS is to increase the volume of the upper airway, predominantly by increasing the volume of the velopharynx, and this increased volume is associated with changes in the surrounding bony and soft tissue structures.