Vernonia cinerea Less. (Asteraceae) or Little ironweed has been demonstrated to possess not only antipyretic and anti-inflammatory effects, but also contain therapeutic activities for alleviating certain gastrointestinal disorders and skin disorders. Trunk is a part of this plant, which is also used as a traditional medicine, but there are no scientific data on anti-inflammatory property in humans. Herein, the present study aimed at evaluating the toxicity of the hexane extract of Vernonia cinerea L. trunk and its anti-inflammatory effect on peripheral blood mononuclear cells (PBMCs) of healthy individuals. Using the MTT assay, we found that the hexane extract did not influence cell viability of PBMCs, and the percentage of viable cells observed was more than 90% with regard to treatment with the concentration range of 0.19 to 100 μg/mL of hexane extract. As experimentally tested using an ELISA method, we found that the hexane extract reduced pro-inflammatory cytokine level (IL-6) significantly (P <0.05) when treating the PBMCs with the concentration range of 12.5 to 100 μg/mL, and this decrease in IL-6 might be due to an inhibition of NF-kB nuclear translocation as proven using immunocytochemistry and confocal microscope. Nevertheless, no changes in TNF-α and IL-10 levels were observed. Therefore, this study suggests that the hexane extract of Vernonia cinerea L. trunk possesses an immunomodulatory effect on human PBMCs.