The mechanism of action on biomolecules of N2 gas plasma, a novel sterilization technique, remains unclear. Here, the effect of N2 gas plasma on protein structure was investigated. BSA, which was used as the model protein, was exposed to N2 gas plasma generated by short-time high voltage pulses from a static induction thyristor power supply. N2 gas plasma-treated BSA at 1.5 kilo pulses per second showed evidence of degradation and modification when assessed by Coomassie brilliant blue staining and ultraviolet spectroscopy at 280 nm. Fourier transform infrared spectroscopy analysis was used to determine the protein's secondary structure. When the amide I region was analyzed in the infrared spectra according to curve fitting and Fourier self-deconvolution, N2 gas plasma-treated BSA showed increased α-helix and decreased β-turn content. Because heating decreased α-helix and increased β-sheet content, the structural changes induced by N2 gas plasma-treatment of BSA were not caused by high temperatures. Thus, the present results suggest that conformational changes induced by N2 gas plasma are mediated by mechanisms distinct from heat denaturation.