Fourier-transform infrared (FT-IR) microspectroscopic mapping can be used to distinguish between different tissue structures, and to increase the image contrast between normal and cancerous regions of a given tissue sample. This study demonstrates the biochemical changes associated with a consistent link between cancerous tissue and various molecular changes in the IR spectra of human pancreatic cancer tissue using FT-IR mapping. Tissue samples were obtained immediately after resection in a patient who underwent a distal pancreatectomy including the pancreatic body and tail. The biochemical imaging changes of lipids, proteins, and nucleic acids in human pancreatic cancer tissue were analyzed via FT-IR microspectroscopy, using imaging, mapping, and line scan techniques. The intensities and frequencies of the absorption bands in the IR spectra of human pancreatic cancerous tissue were markedly reduced and shifted, particularly in the amide bands of protein and CH2 and CH3 stretching vibrations of lipids. The cancerous tissue contained significant protein content, and the distributions of DNA and lipid were very low, indicating low amounts of nucleic acids and lipids in human pancreatic cancer tissue. The analytical results indicate that these FT-IR microspectroscopic biochemical images reflect the distribution of cell components, which could be correlated with stained tissue in adenocarcinoma in pancreatic tissues. This study with samples of noncancerous and cancerous pancreatic tissues has clearly demonstrated that FT-IR microspectroscopy using the mapping method can be used for diagnosis.