Isothermal compression tests were conducted to investigate the hot deformation behaviour of a Ti-6Al-4V alloy with different hydrogen contents (0, 0.35 and 0.6wt.%) at temperatures of 1050-1100°C, and strain rates of 0.005, 0.01, 0.1 and 1s-1 respectively. The microstructural evolution was investigated via optical microscope (OM), X-ray diffraction (XRD) and transmission electron microscope (TEM). The mechanism of hydrogen-induced hardening was discussed. The experimental results showed that hydrogen could be retained in Ti-6Al-4V alloy even though the temperature was increased to 1100°C in air. δ hydride with an face-centred cubic (FCC) crystal structure existed in the deformed matrix, and the size of δ hydride reduced when the deformation temperature was increased from 1050 to 1100°C. Hydrogen induced the increased flow stress and work hardening rate of Ti-6Al-4V alloy when deformed in β phase field. Hydrogen had a positive effect on the development of twinning in Ti-6Al-4V alloy. Based on the analysis of both hot deformation behaviour and microstructural evolution, it is indicated that the hydrogen-induced twinning plays a key role in the enhancement of work hardening of Ti-6Al-4V alloy in β phase field.