AbstractLeishmania donovani, a protozoan parasite, resides and replicates in macrophages and inflicts the potentially fatal disease visceral leishmaniasis (VL). The parasite‐expressed surface lipophosphoglycan (LPG) was implicated in binding TLR2 on NK cells, but the modus operandi of its disease‐promoting influence remained unknown. As TPL2, a member of the MAPK module in mammalian macrophages, was implicated in the anti‐inflammatory immune response and promoting pathogen survival, we investigated the possibility of TPL2‐directed LPG‐TLR2 signalling in Leishmania infection. We observed that TLR2 or TPL2 blockade differentially influenced the TLR2 ligand proteoglycan (PGN)‐induced p38MAPK and ERK‐1/2 activation. TLR2 blockade abrogated the PGN‐induced TPL2 activation. L. donovani infection impaired the Akt activation whereas, upon TPL2 inhibition, the infection fails to control Akt phosphorylation. In L. donovani‐infected macrophages, TLR2 blocking negatively affected p38, Akt and TPL2 phosphorylation while ERK1/2 phosphorylation increased relative to the infection alone. TPL2 blockade reduced TGF‐β, but increased TNF‐α expression and diminished amastigote count in macrophages. While exploring stimulation patterns of TLR2 ligands, LPG, unlike PGN, selectively increased TLR2 expression in macrophages. LPG blockade increased p38MAPK and AKT, but slightly affected ERK‐1/2 and significantly reduced TPL2 phosphorylation from L. donovani‐infected macrophages. Molecular docking and molecular dynamics analysis drew a parallel between LPG's glycan chain lengths with the frequency of interaction with TLR2 which might impact TLR2 signalling. Therefore, the parasite regulates the TLR2 signalling via TPL2 when elicited by LPG‐TLR2 interaction for pathogenesis.