To investigate whether single-nucleotide polymorphisms (SNPs) in toll-like receptors (TLR) 2, 4 and 5 affect the susceptibility of Legionella pneumophila infection in a Han Chinese population by in vitro assay.Fifty-four (n = 54) healthy subjects were genotyped for SNPs (TLR2 (C597T) [rs3804099], TLR4 (G2244A), TLR4 (A2299G) [AF177765] and TLR5 (C1174T) [rs5744168]). Peripheral blood mononuclear cells (PBMCs) were obtained from these subjects and stimulated with live L. pneumophila for 24 h. The mRNA expression levels of adapter protein myeloid differentiation factor 88 (MyD88) were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and the expression levels of TNF-α, IL-1β and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA).After 24 h of L. pneumophila stimulation, the mRNA expression level of MyD88 was significantly lower with TLR2 (C597T) CT/TT (p = 0.0482) or TLR5 (C1174T) CC homozygotes (p = 0.0223) in comparison to PBMCs with other genotypes. The mRNA expression level of MyD88 was significantly higher with TLR4 (G2244A) GG/GA than AA homozygotes (p = 0.0352). No significant difference was found in PBMCs with genotypes TLR4 (A2299G) AA, AG or GG (p > 0.05). Supernatant from cultures of PBMCs with genotype TLR2 (C597T) CT/TT or TLR5 (C1174T) CC were found to have higher levels of TNF-α, IL-1β and IL-6 after L. pneumophila stimulation. TLR4 (G2244A) GG/GA alleles were found to have lower levels of TNF-α (p = 0.0367) and higher levels of IL-6 (p = 0.0317) in comparison to cells with AA alleles. No significant association was observed between the TNF-α, IL-1β and IL-6 levels and genotype TLR4 (A2299G) after L. pneumophila stimulation (p > 0.05).Our findings suggested that healthy subjects who were positive for the TLR2 (C597T) CT/TT and TLR5 (C1174T) CC alleles had a superior innate immune response to L. pneumophila than other genotypes in the evaluated Han Chinese population, whereas no association was found for the TLR4 (A2299G) [AF177765] polymorphism with L. pneumophila susceptibility. It is not clear from our study if TLR4 (G2244A) [AF177765] is associated with susceptibility to L. pneumophila infection.