A species-specific ascaroside-type glycolipid was identified in the nematode Caenorhabditis nigoni using HPLC-ESI-(-)-MS/MS precursor ion scanning, HR-MS/MS, and NMR techniques. Its structure containing an l-3,6-dideoxy-lyxo-hexose unit was established by total synthesis. The identification of this novel 4-epi-ascaroside (caenorhabdoside) in C. nigoni along with the previous identification of 2-epi-ascarosides (paratosides) in Pristionchus pacificus indicate that nematodes can generate highly specific signaling molecules by epimerization of the ascarylose building block downstream of the canonical β-oxidation cycle.