The evolution of the ω phase and its role in plastic deformation in a twinning-induced plasticity β Ti–12 V–2Fe–1Al alloy aged at 373–573 K have been studied in this work. The results show that isothermal ω forms at about 398 K and above, and the growth of isothermal ω is accompanied by a rejection of solute atoms from ω into β. The composition partitioning increases internal elastic strain energy and hydrostatic pressure resulting from the coherency of isothermal ω phase in β. Moreover, the isothermal ω phase becomes hard and even undeformable due to composition partitioning based on first-principles calculations. It is suggested that the internal elastic strain stabilizes the β phase, which suppresses {332} deformation twinning and leads to dislocation glide and stress-induced ω phase transformation in the alloy aged at low temperature. And the embrittlement of the alloy aged at high temperature is thought to arise from undeformability of isothermal ω phase. Display omitted •Evolution of ω phase and its role in plastic deformation in aged β Ti–12 V–2Fe–1Al alloy were investigated.•Isothermal ω forms at 398 K and growth of ω is accompanied by rejecting solute-atoms from ω into β.•Composition partitioning increases internal elastic strain energy and hydrostatic pressure due to coherency of ω in β.•Internal elastic strain stabilizes β phase and changes plastic deformation mechanism of aged alloy.•Isothermal ω becomes hard and even undeformable due to composition partitioning, leading to brittle fracture of aged alloy.