This study evaluated the effect of heating methods on alteration of protein structure in flaxseed (Linum usitatissimum L.) in relation to changes in protein subfraction profile and digestion in dairy cows. Seeds from 2 flaxseed varieties, sampled from 2 replicate plots at 2 locations, were evaluated. The plots (n = 4) were used as replicates. The seeds were either maintained in their raw state or heated in an air-draft oven (dry heating) or autoclave (moist heating) for 60 min at 120°C or by microwave irradiation (MIR) for 5 min. Compared with raw seeds, moist heating decreased (P < 0.05) soluble protein (SP) content (56.5 ± 5.55 to 25.9 ± 6.16% crude protein) and increased (P < 0.05) rumen undegraded protein (RUP) content (36.0 ± 5.19 to 46.9 ± 2.72% CP) and intestinal digestibility of RUP (61.0 ± 2.28 to 63.8 ± 2.67% RUP). Dry heating did not alter (P > 0.05) the protein subfraction profile and rumen degradation kinetics, whereas MIR increased (P < 0.05) the RUP content from 36.0 ± 5.19 to 40.4 ± 4.67% CP. The MIR and dry heating did not alter (P > 0.05) the amide I to amide II ratio, but moist heating decreased (P < 0.05) both the amide I to amide II ratio and α-helix-to-β-sheet ratio. Regression equations based on protein molecular spectral intensities provided high prediction power for estimation of heat-induced changes in SP (R² = 0.62), RUP (R² = 0.71), and intestinal digestibility of RUP (R² = 0.72). Overall, heat-induced changes in protein nutritive value and digestion were strongly associated with heat-induced alteration in protein molecular structures.