SUMMARY Sphingolipids are crucial components of cell membranes. Sphingolipid Δ8-unsaturation is more specific to plants and is involved in the regulation of stress responses. The structure and functions of sphingolipids in microalgae are still poorly understood. Ostreococus tauri is a minimal microalga at the base of the green lineage, and is therefore a key organism for understanding lipid evolution. The present work reports the characterisation as well as the temperature regulation of sphingolipids and Δ8-sphingolipid desaturase from O. tauri . Complex sphingolipids are glycosylceramides with unique glycosyl moieties encompassing hexuronic acid residues, reminiscent of bacterial glucuronosylceramides, with up to three additional hexose residues. In contrast, the ceramide backbones show limited variety, with dihydroxylated C18/C18:1 EΔ8 sphingoid bases and C16:0 fatty-acyl chain being the main compounds. The sphingolipid Δ8-desaturase from O. tauri , although phylogenetically related to plant homologues has a substrate preference similar to the diatom homologue. Both sphingolipid Δ8-desaturase transcripts and sphingolipid Δ8-unsaturation are regulated in a temperature- dependent manner being higher at 14°C than 24°C. Overexpressing the sphingolipid Δ8- desaturase in O. tauri at 24°C results in higher sphingolipid unsaturation and impairs the increase in cell size, structure and chlorophyll. In particular, the cell-size defect is not detected in cells acclimated to 14°C and is furthermore suppressed upon transfer from 24°C to 14°C. Our work provides the first functional evidence for the involvement of sphingolipid Δ8-unsaturation for temperature acclimation in microalgae, suggesting that this function is an ancestral feature in the green lineage.