Arsenic trioxide (ATO) is one of the most potent drugs in cancer chemotherapy, and is highly effective in treating both newly diagnosed and relapse patients with acute promyelocytic leukemia (APL). Despite a number of reports regarding the molecular mechanisms by which ATO promotes anti-tumor or pro-apoptotic activity in hematological and other solid malignancies, the effects of ATO on immune responses remain poorly understood. To further understand and clarify the effects of ATO on immune responses, we sought to examine whether ATO affects the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, RAW 264.7. Arsenic trioxide was found to prevent NO production in a dose-dependent manner. Arsenic trioxide significantly inhibited the increase in inducible nitric oxide synthase (iNOS) at both the mRNA and protein levels. Furthermore, our analyses revealed that the inhibitory effect of ATO on iNOS expression was ascribed to the prevention of IRF3 phosphorylation, interferon (IFN)-β expression, and STAT1 phosphorylation, but not the prevention of the MyD88-dependent pathway. Taken together, our results indicate that ATO prevents NO production by inhibiting the TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-dependent pathway, thus highlighting an anti-inflammatory property of ATO in innate immunity.