•Effect of thawing method on thermal stability and structure of protein was analyzed.•Change in thermal stability of MP induced by thawing method was significant.•Change in structure of protein induced by different thawing methods was demonstrated.•Compared to FM, change in thermal stability and structure from VT was insignificant. This study investigated the effects of thawing methods on the thermal stability and structure of myofibrillar protein (MP) from porcine longissimus dorsi. DSC was used to evaluate the thermal stability (Tmax, △H) of MP. FT-IR, Raman, UV absorption, and fluorescence spectra were utilized to assess the secondary and tertiary structures of MP. Changes in the thermal stability and structure of MP after thawing were significant (P < 0.05), except for the vacuum thawing (VT) samples. The lowest Tmax and △H were obtained through microwave thawing (MT). The decreases in α-helices, β-sheets, fluorescence intensity, and total sulfhydryl content, and increases in the intensity of the SS stretching band and Ca2+-ATPase activity illustrated that secondary structure destruction, tertiary structure unfolding, and disulphide bond cross-linking occurred during thawing. The thawing process caused thermal stability degradation and structure destruction; the largest changes in all indexes of MP were obtained through MT.