Silurus asotus (Amur catfish) egg lectin (SAL) inhibits cell proliferation and enhances the effects of anticancer drugs by binding to globotriaosylceramide (Gb3) on the cell surface. Gb3 expression is typically increased in seminomas. However, its association with SAL and the underlying mechanisms remain unclear. Here, we investigated the effects of SAL on morphology, migratory ability, and integrin expression in JKT-1 cells using chromatography and mass spectrometry. Gb3 was expressed in JKT-1, an established seminoma cell line. SAL did not alter JKT-1 proliferation but increased propidium iodide uptake. Furthermore, SAL induced morphological changes and increased the expression of integrin α2 in JKT-1, but not in HeLa cells. Gb3 expression was detected in JKT-1 and HeLa cells, with high- and low-mobility bands observed. However, the low-mobility bands were more abundant in JKT-1 than in HeLa cells. The main forms of Gb3 in JKT-1 cells were high-mobility d18:1/24:0 and d18:1/24:1 and low-mobility hydroxylated Gb3. Fatty acid 2-hydroxylase was involved in the acyl chain hydroxylation of low-mobility Gb3 in JKT-1 cells and showed 5-fold higher expression in JKT-1 cells than in HeLa cells. Our findings suggest that the antitumor effects of SAL vary according to the specific Gb3 molecular species expressed in cancer cells.