MNSF is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSF covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSF in macrophage cell line, Raw 264.7 cells. We present evidence that MNSF/Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSF/Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSF siRNA LPS-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA mediated knockdown of MNSF increased tumor necrosis factor (TNF) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSF expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNF production. Co-transfection experiments with MNSF and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSF might be implicated in the macrophage response to LPS.