Glycosylceramides are ubiquitous and important components of the plasma membrane in most eukaryotic cells and a few bacteria. They play significant roles in a variety of cellular functions. Their molecular structures are well recognized in animals, higher plants, and fungi, but are poorly characterized in lower plants. In this study, a high glycosylceramide-producing microalgal strain Tetraselmis sp. NKG 400013 was found. TLC and MS analyses established the presence of glycosylceramides, GT1 and GT2, in this strain. Their chemical structures were determined by NMR spectroscopy and GC/MS, and were identified as glycosylceramides consisting of the typical botanical sphingoid base ([4E, 8E]-sphinga-4, 8-dienine) and 2-hydroxy-Δ3-unsaturated fatty acyl chains, respectively. To our knowledge, the occurrence of glycosylceramides in microalga of the class Prasinophyceae was previously unknown.