This research is focused on a study of the relationship between mechanical behavior, microstructure and fracture toughness of a Ti-6Al-4V alloy in both coarse-grained (CG) and ultrafine-grained (UFG) conditions. The UFG state with a primary alpha-phase grain size, but with different orientations with respect to the testing direction, was produced by equal-channel angular pressing (ECAP) after thermo-mechanical treatment. Fracture toughness and mechanical testing were conducted at a temperature of − 196 °C. A duplex UFG structure formation in the Ti-6Al-4V alloy led to an enhancement of yield stress and a decrease in the fracture toughness at − 196 °С by comparison with the CG alloy. The lowest values of fracture toughness were observed in a sample in which there were elongated grains lying parallel to the loading direction during testing compared to the situation where the grains were perpendicular to this direction. The reasons for the reduction in fracture toughness in the UFG Ti alloy are discussed.