RhoA, an extensively studied member of the Rho GTPase family, has been identified as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Bacterial lipopolysaccharide (LPS) is known to be a potent stimulator of inflammatory cytokine production. LPS is able to alter the activity of RhoA and the subcellular distribution of RhoA is altered according to its activity. In this study, we investigated a possible link between RhoA and the LPS/nuclear factor (NF)-κB signaling pathway. In the present study, western blotting and pull-down and immunofluorescence assays were performed to investigate the activity of RhoA in A549 cells following LPS stimulation. The results showed that LPS was able to activate RhoA. Furthermore, western blotting and an immunofluorescence assay were carried out to investigate the nuclear expression of RhoA in A549 cells following LPS stimulation. The results indicated that LPS triggers the nuclear translocation of RhoA. Furthermore, western blotting, NF-κB small interfering RNA (siRNA) transfection and an immunofluorescence assay were performed to investigate the role of NF-κB in LPS-induced RhoA nuclear translocation in A549 cells. The results showed that LPS-induced RhoA nuclear translocation was inhibited by NF-κB depletion in A549 cells. RhoA and NF-κB siRNA transfection, western blotting and ELISA were carried out to investigate the role of RhoA in the LPS-induced secretion of interleukin (IL)-6 and IL-8 in A549 cells. The depletion of RhoA using RhoA siRNA decreased the LPS-induced secretion of IL-6 and IL-8, similar to the effect of NF-κB depletion. These results demonstrate that LPS is able to activate RhoA and trigger its nuclear translocation, which is dependent on NF-κB, and that RhoA plays a significant role in the LPS/NF-κB signaling pathway.