The cytosolic nucleotide-binding oligomerization domain 1 (NOD1)/CARD4 and NOD2/CARD15 proteins are members of NOD-like receptors recognizing specific motifs within peptidoglycans of both Gram-negative and Gram-positive bacteria. NOD1 and NOD2 signal via the downstream adaptor serine/threonine kinase RIP2/CARDIAK/RICK to initiate NF-κB activation and the release of inflammatory cytokines/chemokines. In this report, we show that 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a cell-permeable, small molecule that has anti-tumor activity, can also activate NOD1 and NOD2. This was demonstrated: 1) by using human embryonic kidney epithelial (HEK) 293 cells transfected with a NF-κB reporter plasmid in combination with NOD1 or NOD2 expression plasmids; 2) by inhibiting DMXAA-induced chemokine (CXCL10) mRNA and protein production in the AB12 mesothelioma cell line using a pharmacological inhibitor of RICK kinase, SB20358; and 3) by using small interfering RNA to knock down NOD2 and lentiviral short hairpin RNA to knock down RICK. These findings expand the potential ligands for the NOD-like receptors, suggesting that other xanthone compounds may act similarly and could be developed as anti-tumor agents. This information also expands our knowledge on the mechanisms of action of the anti-tumor agent DMXAA (currently in clinical trials) and may be important for its biological activity.