The Tenebrio molitor larvae protein isolate (TPI) was first extracted via alkaline solubilization and acid precipitation. Subsequently the effects of heat treatment and dynamic high-pressure microfluidization on the physicochemical, functional properties and the in vitro bioaccessibility of the TPI were investigated. The results showed that the higher order structure of native TPI was mainly stabilized through hydrophobic interaction, hydrogen bonds, and disulfide bonds. TPI aggregates could be induced by both heat and microfluidization, of which the aggregation was possibly dominated by hydrophobic interactions. The solubility of the TPI decreased slightly after heat treatment, likely due to the exposure of the hydrophobic groups inside the protein impairing the interaction between the TPI and water molecules. The mechanical force of microfluidization improved the interaction between the TPI and water molecules and distinctly increased TPI solubility, which was positively correlated with the pressure level. Under neutral conditions, the solubility of the TPI after microfluidization at 50 and 100 MPa was elevated from 24.05% to 50.08% and 64.81%, respectively. The emulsifying and foaming properties of the TPI were improved by heat treatment, although this weakened the emulsifying stability. Microfluidization increased the foaming stability of the TPI yet had no considerable impact on its emulsifying properties and foaming capacity. Both types of physical treatments increased the in vitro bioaccessibility of the TPI, since the nitrogen releases of 90 ℃ and 120 ℃ heat-treated TPI were reduced from 39.53 % to 35.58% and 33.90%, respectively. Furthermore, the TPI subunits at approximately 17 ku could not be hydrolyzed thoroughly after microfluidization.