Lipomannans (LM) are powerful pro-inflammatory lipoglycans found in mycobacteria and related genera, however the molecular bases of their activity are not fully understood. We report here the isolation and the structural and functional characterization of a new lipomannan variant present in the Pseudonocardineae, Saccharothrix aerocolonigenes, designated SaeLM. Using a range of chemical degradations, NMR experiments and mass spectrometry analyses, SaeLM revealed a mannosyl-phosphatidyl-myo-inositol (MPI) anchor glycosylated by an original carbohydrate structure whereby (a1-6)-Manp backbone is substituted at more than 80% of the O-2 position by side chains composed of Manp-(a1-2)-Manp-(a1-. MALDI-MS analysis indicated a distribution of SaeLM glyco-forms ranging from 19 to 61 Manp units, which centred on species containing 37 or 40 Manp units. SaeLM induced a TLR-2-dependant production of TNF-a by human THP-1 monocyte/macrophage cell lines and interestingly was found to be the strongest inducer of this pro-inflammatory cytokine when compared to other LAM/LM-like molecules. We previously established that a linear (a1-6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity. We demonstrate here that by adding side chains and increasing their size, one may potentiate this activity. These findings should enable a better understanding of the structure/function relationships of TLR-2 dependent lipoglycan signaling.