Monoclonal nonspecific suppressor factor β (MNSFβ) is a ubiquitously expressed ubiquitin-like protein known to be involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently modify its target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 65 kDa MNSFβ adduct from mouse liver lysates by sequential chromatography on DEAE and glutathione S-transferase (GST)-fusioned MNSFβ immobilized on glutathione-Sepharose beads in the presence of ATP. MALDI-TOF mass spectrometry fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and heat shock protein 60 (HSP60), a mitochondrial protein involved in protein folding. Fingerprinting analysis of the MNSFβ adduct demonstrates that MNSFβ conjugates to HSP60 with a linkage between the C-terminal Gly74 and Lys481. HSP60 siRNA neutralized the inhibition of apoptosis by MNSFβ siRNA in LPS/IFNγ-stimulated Raw264.7, a murine macrophage cell line. HSP60 siRNA also down-regulated the enhancement of TNFα production by MNSFβ siRNA in LPS-stimulated Raw264.7 cells. Here, we firstly report that MNSFβ activity is negatively regulated by molecular chaperone.