Distribution of 9-O-acetylated sialic acids (9-O-AcSA) on Leishmania donovani has been previously reported. Considering their role in recognition, the differential distribution of sialic acids especially 9-O-acetylated sialic acids in avirulent (UR6) versus virulent (AG83 and GE1) promastigotes of Leishmania donovani and its role in entry into macrophages was explored. Fluorimetric-HPLC, fluorimetric determination and ELISA revealed 14-, 8- and 5-fold lower sialic acids in UR6 as compared to AG83. Interestingly, on UR6, flow cytometry indicated lower (alpha2-->6)-linked sialoglycoproteins along with minimal 9-O-acetylated sialoglycoproteins by Scatchard analysis. Further, UR6 demonstrated a 9- and 14.5-fold lower infectivity and phagocytic index than AG83. Additionally, de-O-acetylation and de-sialylation of AG83 demonstrated a 3- and 1.5-fold reduced phagocytic index. The role of 9-O-AcSA in entry was further confirmed by pre-blocking the macrophage surface with a cocktail of sugars followed by microscopic quantification. The phagocytic index of AG83 exclusively through 9-O-AcSA was significantly high. Interestingly, AG83 produced higher metacyclic promastigotes containing increased 9-O-AcSA as compared to avirulent UR6 supporting its virulent nature. Taken together; our results conclusively demonstrate the increased presence of 9-O-acetylated sialic acid on promastigotes of virulent Leishmania donovani as compared to avirulent UR6 and their subsequent role in entry within macrophages.