Glycosphingolipids are ubiquitous cell membrane and act significantly in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. The advances of structural analysis of glycosphingolipids using modern analytical instruments, the separation and identification of glycosphingolipids, as well as the biological functions of glycosphingolipids in the progress of diseases were thoroughly discussed in this review. The analysis of glycosphingolipid has made spectacular progress due to the rapid development of modern analytical technology, especially mass spectrometry and the tandem application of chromatography-mass spectrometry. Presently, the use of mass spectrometry in discovering aberrant levels of multiple glycosphingolipids in various malignancies has indicated that the fucosylated glycosphingolipids elevated in the tumor tissues of hepatocellular carcinoma, colorectal cancer, breast cancer and so on. This phenomenon made fucosylated glycosphingolipid a potentially novel cancer marker for early diagnosis. Furthermore, phenotypic and functional research on glycosphingolipids is becoming a popular aspect of glycolipid study, particularly in the relevancy between glycosphingolipids and angiogenesis. For instance, glycosphingolipids shed from the surface of tumor cells act positively on angiogenesis, nevertheless, GM3, a simple ganglioside, acts reversely as a proangiogenesic factor in the tumor microenvironment. This review summarized several cellular hypothesis of the phenomenon above, illustrated the explorations in glycosphingolipid-targeted therapy against malignancies to provide further visions on glycosphingolipid research.