To date there has been very little application of synchrotron radiation-based Fourier transform infrared microspectroscopy (SRFTIRM) to the study of molecular structures in plant forage in relation to livestock digestive behavior and nutrient availability. Protein inherent structure, among other factors such as protein matrix, affects nutritive quality, fermentation and degradation behavior in both humans and animals. The relative percentage of protein secondary structure influences protein value. A high percentage of beta-sheets usually reduce the access of gastrointestinal digestive enzymes to the protein. Reduced accessibility results in poor digestibility and as a result, low protein value. The objective of this study was to use SRFTIRM to compare protein molecular structure of alfalfa plant tissues transformed with the maize Lc regulatory gene with non-transgenic alfalfa protein within cellular and subcellular dimensions and to quantify protein inherent structure profiles using Gaussian and Lorentzian methods of multi-component peak modeling. Protein molecular structure revealed by this method included alpha-helices, beta-sheets and other structures such as beta-turns and random coils. Hierarchical cluster analysis and principal component analysis of the synchrotron data, as well as accurate spectral analysis based on curve fitting, showed that transgenic alfalfa contained a relatively lower (P<0.05) percentage of the model-fitted alpha-helices (29 vs. 34) and model-fitted beta-sheets (22 vs. 27) and a higher (P<0.05) percentage of other model-fitted structures (49 vs. 39). Transgenic alfalfa protein displayed no difference (P>0.05) in the ratio of alpha-helices to beta-sheets (average: 1.4) and higher (P<0.05) ratios of alpha-helices to others (0.7 vs. 0.9) and beta-sheets to others (0.5 vs. 0.8) than the non-transgenic alfalfa protein. The transgenic protein structures also exhibited no difference (P>0.05) in the vibrational intensity of protein amide I (average of 24) and amide II areas (average of 10) and their ratio (average of 2.4) compared with non-transgenic alfalfa. Cluster analysis and principal component analysis showed no significant differences between the two genotypes in the broad molecular fingerprint region, amides I and II regions, and the carbohydrate molecular region, indicating they are highly related to each other. The results suggest that transgenic Lc-alfalfa leaves contain similar proteins to non-transgenic alfalfa (because amide I and II intensities were identical), but a subtle difference in protein molecular structure after freeze drying. Further study is needed to understand the relationship between these structural profiles and biological features such as protein nutrient availability, protein bypass and digestive behavior of livestock fed with this type of forage.