Several new glycosphingolipids were isolated by means of reversed-phase HPLC from the less-polar fraction of the methanol extract of the sponge Aplysinella rhax. Their structures, showing very close similarity, were elucidated by extensive NMR experiments including 1H,1H (COSY, TOCSY and ROESY) and 1H,13C (HMQC and HMBC) spectroscopy and chemical evidence. The molecular masses of the glycosphingolipids were determined by negative-ion fast-atom bombardment mass spectrometry, while HREIMS and HRFABMS provided important information for the identification of the fatty acids and sphingoid bases. The mixture of glycosphingolipids exhibited a good inhibitory activity on LPS-induced NO2− release by J774 A.1 macrophages.