Thymol is one of the most important dietary constituents in the thyme species and has been shown to possess anti-inflammatory properties both in vivo and in vitro. We investigated the protective effects of thymol on the lipopolysaccharide (LPS)-induced inflammatory responses in the human peritoneal mesothelial cell line (HMrSV5) to clarify the potential mechanism. HMrSV5 cells were stimulated with LPS in the presence or absence of thymol. Our results showed that thymol markedly suppressed the production of cytokines such as tumour necrosis factor α (TNF-α), interleukin (IL)-6, monocyte chemoattractant protein 1 (MCP-1) and α-smooth muscle actin (α-SMA) in a dose-dependent manner. Western blot analysis indicated that RhoA and ROCK activation; Toll-like receptor 4 (TLR4) expression; and Nuclear factor -kappa B (NF-κB) p65, IKK and IκBα phosphorylation were also inhibited by thymol. Moreover, siRNA knockdown of RhoA suppressed the expression of pro-inflammatory cytokines and phosphorylation of NF-κB p65 and IκBα proteins in LPS-stimulated HMrSV5 cells, but did not affect TLR4 expression. In conclusion, thymol inhibits LPS-induced inflammation in HMrSV5 cells by suppressing TLR4-mediated RhoA-dependent NF-κB signalling pathway. Our study suggests that thymol may be a promising therapeutic agent against peritonitis.