In this study, active interpass cooling using various compressed CO2 gas strategies was applied to wire-arc additively manufactured Ti–6Al–4V alloy, and as-manufactured microstructure characteristics, including phase composition, grain growth and microhardness, were fully explored by using Electron Backscattered Diffraction and hardness tester. Results show that rapid interpass cooling brings obvious martensite phase transformation, primarily from lamellar α grains to acicular α grains, to the deposited component. The microstructure tends to be fine-grained, disordered and displays reduced texture strength and increased dislocation density through visible coloration. This feature also exhibits high hardness values that brings an improvement in mechanical strength to deposited metal. Moreover, it is found cooling time variation could obtain better grain refinement than altering cooling gas flow rate during deposit, which is probably related to effective heat dissipation. The research findings can provide significant insight into microstructural evolution mechanism in wire-arc directed energy deposition process, and benefits to properties control.