Interleukin-10 (IL-10) is an important anti-inflammatory cytokine that plays a critical role in the control of immune responses. We previously reported that the time-dependent involvement of the transiently activated cyclin-dependent kinase 5 (CDK5)-p35 complexes suppress various mitogen-activated protein kinases (MAPKs), thereby preventing macrophages from the premature production of suppressor of cytokine signaling 3 (SOCS3) and IL-10 in the early time. However, the direct binding partners between CDK5/p35 and MAPK for new therapeutic targets have not found yet. Therefore, we performed immunoprecipitation of CDK5 as compared with treatment of roscovitine, a CDK5 inhibitor in LPS stimulated murine bone marrow-derived macrophages. Interestingly, 105 candidate down-regulated proteins were gained by mass spectrometry with roscovitine treated group. Among them, we selected G protein alpha inhibitor 3 (GNAI3), one of the target molecules that have CDK5 consensus sequence and the relation with MAPK pathway by using bioinformatics resource. Furthermore, the knockdown of GNAI3 using siRNA significantly increased IL-10 production in LPS activated macrophages (p<0.05). Collectively, these findings provide support for the future development and clinical application of GNAI3 regulating IL-10 production.