Current cancer treatments destroy the tumor mass but cannot prevent the recurrence of cancer. The heterogeneous structure of the tumor mass includes cancer stem cells that are responsible for tumor relapse, treatment resistance, invasion and metastasis. The biology of these cells is still not fully understood; therefore, effective treatments cannot be developed sufficiently. Herein, attenuated total reflection- Fourier transform infrared (ATR-FTIR) spectroscopy, combined with unsupervised multivariate analysis, was applied to prostate cancer stem cells (CSCs), non-stem cancer cells (non-CSCs) and normal prostate epithelial cells to elucidate the molecular mechanisms and features of CSCs, which are crucial to improving the target specific therapies. This work revealed the spectral differences in the cellular mechanisms and biochemical structures among three different cell types. Particularly, prostate CSCs exhibit differences in the lipid composition and dynamics when compared to other cell types. CSCs also harbor pronounced differences in their major cellular macromolecules, including differences in the protein amount and content (mainly a-helices), the abundance of nucleic acids (DNA/RNA), altered nucleic acid conformation and carbohydrate composition. Interestingly, macromolecules containing the CvO groups and negatively charged molecules having the COO-groups are abundant in prostate CSCs in comparison to prostate non-CSCs and normal prostate cells. Overall, this study demonstrates the potential use of ATR-FTIR spectroscopy as a powerful tool to obtain new insights into the understanding of the CSC features, which may provide new strategies for cancer treatment by selectively targeting the CSCs.