Cellular macromolecules play important roles in cellular behaviors and biological processes. In the current work, cancer (KLN205), normal (MSFs) and mouse embryonic stem cells (mESCs) are compared using ATR-FTIR spectroscopy. Modifications in the composition, concentration, structure and function-related changes in the cellular components were deciphered using the infrared spectra. Our results revealed that cancer and embryonic stem cells are very similar but highly different from the normal cells based on the spectral variations in the protein, lipid, carbohydrate and nucleic acid components. The longest lipid acyl chains exist in mESCs, while cancer cells harbor the lowest lipid amount, short lipid acyl chains, a high content of branched fatty acids and thin cell membranes. The highest cellular growth rate and accelerated cell divisions were observed in the cancer cells. However, the normal cells harbor low nucleic acid and glycogen amounts but have a higher lipid composition. Any defect in the signaling pathways and/or biosynthesis of these cellular parameters during the embryonic-to-somatic cell transition may lead to physiological and molecular events that promote cancer initiation, progression and drug resistance. We conclude that an improved understanding of both similarities and differences in the cellular mechanisms among the cancer, normal and mESCs is crucial to develop a potential clinical relevance, and ATR-FITR can be successfully used as a novel approach to gain new insights into the stem cell and cancer research. We suggest that targeting the cellular metabolisms (glycogen and lipid) can provide new strategies for cancer treatment.