The microstructures of Ti6Al4V alloy after hydrogenation have been investigated and analysed by optical microscopy (OM), X-ray diffraction (XRD) and transmission electron microscopy (TEM), and the influence of hydrogenation on the hardness of α and β phases has been analysed by microhardness testing. The microstructure observation revealed that δ hydride (fcc structure) precipitated in the specimens with 0.278 wt.% and 0.514 wt.% hydrogen, and a lot of dislocations and twins have been found simultaneously. The diffraction peaks moved to the lower angles because of the lattice expansion of β phase with the solution of hydrogen atoms. The result of microhardness testing shows that the hardness of α and β phases increases synchronously with increasing of hydrogen and the increment of β is larger than that of α. It is considered that the formation of δ hydrides, lattice defects and alloying element diffusion are the major factors leading to the microhardness change.