Streptococcus pneumoniae (SPN) is the single most deadly bacterial infection globally, capable of causing a wild spectrum disease, including sinusitis, otitis media, pneumonia, bacteremia and meningitis. The mortality caused by pneumococcal disease is sometimes linked to imbalanced hyper-inflammatory responses of the host despite of successful pathogen clearance. Immune cell surfaces are richly equipped with various lectins and glycans by which they use to sense and process the external information and to elicit integrated intracellular signaling cascade and appropriate immune responses. We previously demonstrated that pneumococcal sialidase (NanA) can provoke leukocyte inflammatory responses through removing the cis-lignads of Siglec expressing on the same myeloid cells. Here, we further explored the underlying molecular mechanisms responsible for this NanA-mediated aggravated inflammation. We found that depletion of cholesterols from lipid rafts partially abrogated NanA-potentiated TNF-α production. In addition, pneumococcal NanA dysregulated the recruitment of TLR-2 to lipid raft and affected the activation of signaling molecules downstream of TLR-2. Moreover, diminished interaction between Siglec and TLR-2 and reduced recruitment of phoaphatase SHP-1 to Siglec was also observed in the wild type SPN but not DNanA mutant-infected cells. Our data suggested that interplay between Siglecs and TLRs may play a critical role in the regulation of host inflammation upon pneumococcal infection.