Three new warm-seasoned corn lines (LM10, LM01 and LD999) were used in this study to reveal mechanism with which protein molecular structure determined protein rumen and intestinal digestion characteristics. Protein molecular structure features were determined by using attenuated total reflectance Fourier-transform vibrational molecular spectroscopy; then revealed by OMNIC in 2 major peaks regions: amide I (ca. 1720–1575 cm⁻¹) and amide II (ca. 1575–1489 cm⁻¹). Protein structural α-helix (centered at ca. 1650 cm⁻¹) and β-sheet (centered at ca. 1640 cm⁻¹) were also derived from Amide I regions. Standard in situ method and 3-step in vitro procedure were applied to evaluate ruminal and intestinal digestion characteristics. PROC MIXED and PROC CORR (SAS 9.4) were applied to analysis of digestibility and molecular spectral features; and Statistica 8.0 was performed to multivariate analyses of molecular spectral data. Molecular spectral intensities of amides I and II and structural α-helix and β-sheet were highest in LD999, and lowest in LM01. Spectral peak height and area ratio of amide I to amide II were both greater in LM01 than the other 2 lines of corn (P < 0.01). Agglomerative hierarchical cluster analysis and principal component analysis results showed that 3 lines of corn could be distinguished from each other in protein molecular spectral region, indicating that they differed in protein molecular features and conformation. Line LD999 had greater crude protein (CP), truly digestible CP and rumen undegradable protein than LM10 and LM01, but rumen degradable protein was greater in LM01 than in LD999. No difference was observed in digestibility of rumen undegradable protein among 3 lines. Total digestible CP was greater in LM10 and LM01 than LD999 (P < 0.01). Correlation analysis showed that protein structural spectral intensity was positively correlated with rumen undegradable protein, but negatively correlated with rumen degradable protein and total digestible CP. Hence, protein molecular structure in warm-seasoned corns apparently influenced protein gastrointestinal digestion characteristics in ruminant animals.