Polo-like kinases (PLKs) have been reported to be essential components of anti-viral pathways. However, the role of PLKs in the production of pro-inflammatory cytokines induced by TLR activation is uncertain. We report here that monocytic THP-1 cells expressed PLK1, PLK2, PLK3 and PLK4. When THP-1 cells were treated with GW843682X, an inhibitor of PLK1 and PLK3, the results showed that GW843682X down-regulated Pam3CSK4- and LPS-induced TNF-α at both the gene and protein levels. GW843682X did not impact Pam3CSK4-induced IL-1β and IL-8 or LPS-induced IL-1β, but it down-regulated LPS-induced IL-8 significantly. Moreover, western blot results showed that TLRs activated PLK1, and PLK1 inhibition by RNA interference down-regulated Pam3CSK4-induced TNF-α production, suggesting the involvement of PLK1 in TNF-α up-regulation. In addition, GW843682X treatment for 12 to 24 h induced cell death and down-regulated MyD88, but neither of these roles contributed to the down-regulation of TNF-α, as TNF-α gene expression was up-regulated at 1 h. Furthermore, GW843682X inhibited Pam3CSK4-induced activation of ERK and NF-κB, which contributed to Pam3CSK4-induced up-regulation of TNF-α. GW843682X also inhibited LPS-induced activation of ERK, p38 and NF-κB, which contributed to LPS-induced up-regulation of TNF-α. Taken together, these results suggested that PLK1 is involved in TLR2- and TLR4-induced inflammation, and GW843682X may be valuable for the regulation of the inflammatory response.