Treatment of chronic osteomyelitis has become a difficult problem in clinical medicine due to its extended pathological process, high occurrence of complication and recurrence of disease. The major pathogenesis mechanism is Gram-negative bacteria infection, such as staphylococci. LPS is an important substance found in the cell wall of Gram-negative bacteria and administration of LPS to skeleton relevant cells in vitro could simulate the pathological characteristics of osteomyelitis patients. The results of quantitative real-time PCR and Western blot showed that CHI3L1 was up-regulated obviously in the infected bone tissues of osteomyelitis patients and LPS-stimulated osteoblasts. Analysis of the luciferase activity of NF-κB reporter gene vector revealed that LPS could activate NF-κB. Bay11-7082, an inhibitor of NF-κB activation, suppressed the elevation of CHI3L1 expression induced by LPS. Pre-incubation of osteoblasts with anti-TNF-α antibody or silencing TNF-α receptor expression by siRNA inhibited the induction effect of LPS on CHI3L1. Inhibition of NF-κB activation also prevented up-regulation of TNF-α induced by LPS. In conclusion, LPS stimulated TNF-α expression through activating NF-κB, then TNF-α induced CHI3L1 expression. It was demonstrated for the first time that CHI3L1 expression is promoted in osteomyelitis and LPS-treated osteoblasts and investigates the molecular mechanism of LPS-induced CHI3L1 expression in osteoblasts.