A plethora of the cancer drugs with high therapeutic potential cannot pass the clinical trials because of their immunotoxic activities. In this study, we tested the immunomodulatory and immunostimulatory effects of the anticancer agent alexidine dihydrochloride on J774.2 macrophage cell lines in vitro. The production levels of the pro-inflammatory cytokines (TNF-α, IL-6, GM-CSF, IL-12p40) were measured and compared by ELISA method. The activated (phosphorylated) JNK protein levels were measured by flow cytometer and the possible related intracellular signaling pathway was examined in this way. According to our results, alexidine dihydrochloride has an anti-inflammatory effect on the LPS-stimulated macrophage cell lines, as evidenced by reduced cytokine production compared to controls. Furthermore, its intracellular mechanism of action was found to be mediated partially through JNK signaling pathways. These findings suggest that alexidine dihydrochloride, while being an effective anticancer agent, may also modulate immune responses by dampening excessive inflammation. In this study, determining the anti-inflammatory effect of alexidine dihydrochloride on the immune system will seriously shed light on the role of this anticancer agent in future clinical studies and will provide a serious basis. In summary, the effects of the most drug-active ingredients on the inflammatory response in immune system cells have not been fully tested, and this creates the problem of many drugs failing in clinical studies or lack of knowledge on their side effects. Our study aimed to determine the effect of alexidine dihydrochloride, used as an anticancer agent, on the inflammatory response in J774.2 macrophage cell lines. Future studies with more immune system cells and a wider analysis of the intracellular signaling pathways will be informative about the immunotoxicity of the drug molecule. Future research involving a broader range of immune cell types and a more comprehensive analysis of intracellular signaling pathways will help clarify the immunotoxicity profile of this anticancer agent.