Sphingolipids have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here we describe the effect of a sphingolipid analogue, Jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line HGC-27. JB induces alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine (dhSo) and sphingosine (So), and their phosphorylated forms, due to inhibition of ceramide synthases (CerS). Moreover, JB induces atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appear to originate from macropinocytosis and trigger cytoplasmic disruption. The pan-caspase inhibitor Z-VAD does not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor wortmannin did not decrease JB-stimulated LC3 II accumulation. In addition, cell vacuolation induced by JB is characterised by single-membrane vacuoles which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on sphingolipid metabolism.