Electron beam welding (EBW) is a fusion joining process particularly suitable for welding titanium plates. In the present work, 2.5 mm thickness Ti6Al4V titanium alloy plates were butt-welded together with backing plates by EBW. The detailed procedures of experiments were used to investigate the microstructure and mechanical properties of welded joints. The optimum welding speed was determined by microstructure examinations, microhardness tests, X-Ray diffraction tests, shear punch tests (SPT) and stress simulation calculations. The results showed that all microstructure of welded metal (WM) was martensite phase under the different welding speeds. In the heat-affected zone (HAZ), the martensite phase gradually evolved to be small and equiaxed. It can be seen that the microstructure of each region in welded joints did not change significantly. When the welding speed is between 8 mm/s and 14 mm/s, it can be seen from the macroscopic appearance of the joints that there was no utterly fused penetration between the butt plate and substrate. Finite element simulation was carried out for the no-penetration depth under different welding conditions, and it was found that the stress suffered by the small no-penetration depth was the smallest. Using different welding parameters shows that the engineering stress in WM was higher than other areas, and BM was the lowest. As welding speed increases from 8 mm/s to 14 mm/s, the variation of microhardness distribution was not evident.