Due to uncontrolled cell proliferation and disrupted vascularization, many cancer cells in solid tumors have limited oxygen supply. The hypoxic microenvironments of tumors lead to metabolic reprogramming of cancer cells, contributing to therapy resistance and metastasis. To identify better targets for the effective removal of hypoxia-adaptive cancer cells, it is crucial to understand how cancer cells alter their metabolism in hypoxic conditions. Here, we studied lipid metabolic changes in cancer cells under hypoxia using coherent Raman scattering (CRS) microscopy. We discovered the accumulation of lipid droplets (LDs) in the endoplasmic reticulum (ER) in hypoxia. Time-lapse CRS microscopy revealed the release of old LDs and the reaccumulated LDs in the ER during hypoxia exposure. Additionally, we explored the impact of carbon sources on LD formation and found that MIA PaCa2 cells preferred fatty acid uptake for LD formation, while glucose was essential to alleviate lipotoxicity. Hyperspectral-stimulated Raman scattering (SRS) microscopy revealed a reduction in cholesteryl ester content and a decrease in lipid saturation levels of LDs in hypoxic MIA PaCa2 cancer cells. This alteration in LD content is linked to reduced efficacy of treatments targeting cholesteryl ester formation. This study unveils important lipid metabolic changes in hypoxic cancer cells, providing insights that could lead to better treatment strategies for hypoxia-resistant cancer cells.