Starfish Asterias amurensis produces sphingoid bases d18:3, 9-methyl-d18:3 (9Me-d18:3), and d22:2, which possess unique structural features. In this study, sphingoid bases prepared from A. amurensis glucosylceramides displayed unexpected elution behaviors from a general octadecyl silyl high-performance liquid chromatography (HPLC) column. For separation and isolation, sphingoid bases were fractionated by octadecyl silyl HPLC after N-acetylation, yielding d18:3, 9Me-d18:3, and two d22:2 isomers. To compare the biological activities of individual sphingoid bases, their effects on sphingolipid production in normal human keratinocytes were evaluated. Treatment with sphingoid bases increased the content of ceramides, glucosylceramides, and sphingomyelins in keratinocytes. Moreover, ceramides, which contain saturated ultra-long-chain fatty acids (C30–34), were significantly increased by treatment with d18:3, but not with other A. amurensis sphingoid bases. The mRNA level of the early differentiation marker keratin 10 was markedly decreased and sphingolipid synthesis-related genes were slightly increased in keratinocytes exposed to A. amurensis-derived d18:3, 9Me-d18:3, and d22:2 isomers. These results suggest that A. amurensis-derived sphingoid bases induce differentiation to varying degrees, sphingolipid production depends on their chemical structures, and d18:3 is the most promising functional sphingoid base.