Receptor-interacting protein 2 (RIP2) is a caspase recruitment domain (CARD)-containing serine/threonine kinase that is activated by NOD1 or NOD2 recognition of their ligands and essential for the activation of NF-κB and mitogen-activated protein kinase (MAPK). RIP2 has been known to play an important role in innate immune responses against certain bacterial infection. However, the role and interplay of RIP2 with TLR signalling on cytokine production in macrophages against Yersinia enterocolitica infection remains poorly understood. In the present study, we examined whether RIP2 is essential for Yersinia-induced production of cytokines in macrophages. Our results showed that naïve RIP2-deficient macrophages produced similar level of IL-6, TNF-α and IL-10 upon Y. enterocolitica infection compared with wild-type macrophages. However, the production of IL-6, TNF-α and IL-10 by Y. enterocolitica was impaired in RIP2-deficient macrophages after lipopolysaccharide (LPS) pretreatment, a TLR4-tolerant condition. In addition, RIP2 inhibitors, SB203580, PP2, and gefitinib, reduced IL-6 production in TLR4-deficient macrophages in response to Y. enterocolitica, whereas they did not affect the cytokines production in WT cells. These results demonstrate that RIP2 may play an important role in proinflammatory cytokine production in macrophages at the absence of TLR signalling.