【Objective】To investigate the effect of pellino-1 gene overexpression by lentivirus vector on the ubiquitin of tumor necrosis factor receptor-associated factors 3（TRAF3）and the activation of mitogen-activated protein kinases（MAPK）signaling pathway in endotoxin-tolerant kupffer cells（KCs）.【Methods】Isolated KCs of C57BL/6 mouse were randomly divided into two groups：control group，which transfected with control lentivirus vector for 48 h，then pretreated with 10 ng/mL lipopolysaccharide （LPS）for 24 h，and next treated with 300 ng/mL LPS；overexpression group，which transfected with pellino-1 gene overexpression lentivirus vector for 48 h，then pretreated with 10 ng/mL lipopolysaccharide（LPS）for 24 h，and next treated with 300 ng/mL LPS. The protein expressions of pellino-1，K48-Ub，TRAF3，p38，p-p38，c-Jun N-terminal kinase（JNK）and p-JNK were analyzed by Western blot. The level of interleukin-1β（IL-1β），tumor necrosis factor-α（TNF-α）and interleukin-10（IL-10）in superna? tants were measured by enzyme linked immunosorbent assay（ELISA）.【Results】Compared with control group，the protein expres? sions of pellino-1，K48-Ub，p-JNK and p-p38 and the levels of IL-1β（P < 0.05），TNF-α（P < 0.05）in supernatants was in?creased in overexpression group，while the protein levels of TRAF3 and the levels of IL-10（P < 0.05）in supernatants were de? creased.【Conclusion】Overexpression of pellino-1 can promote TRAF3 K48 ubiquitination degradation，decrease the protein expres? sion of TRAF3，activate the downstream MAPK signaling pathway，and thus impair the formulation of endotoxin tolerance.