In order to explore the capability of Fourier transform infrared microspecroscopy in discriminating single gene difference in single cell, synchrotron Fourier transform infrared microspecroscopy is used to measure single cell spectra of colorectal cancer cells with and without tumor suppressor gene p53, respectively. With comparison of spectra of these two types of cancer cells, absorption intensities and frequencies of lipids, proteins and nucleic acids are evidently different. The absorption bands of single cell without p53 are weak and almost all the peaks drift to higher wavenumbers. Absorption intensity ratio of amide I/amide II increases significantly in the cell without p53, indicating the secondary structures of proteins change. The fitting analysis of amide I absorption band reveals that contents of α helix and random coil in secondary structures of proteins in cell with p53 gene are lower than that in cell without p53, and contents of turns and atypical helix are higher in cell with p53. However, content of β-sheet in both cell is very similar. The results demonstrate that the absence of single gene p53 can affect metabolism activity, which can be clarified by synchrotron Fourier transform infrared microspecroscopy at molecular level.