The PET-28a(+)expression plasmids were constructed according to the identified HMGB1 homologous gene sequences (CDSs)of Clonorchis sinensis and cloned into Rosetta (DE3) competent cells. Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of CsHMGB1-His fusion proteins. After purification by Ni-TED agarose resin, the expression of His-tagged fusion proteins was confirmed by Western blotting analysis. The mouse macrophage RAW264.7 cells were cultured with mediums supplemented with CsHMGB1-159/127 at a concentration of 1 μg/ml and 10 μg/ml, for 24 h and 48 h, respectively. The cells cultured without stimulants were used as the blank control. The SEAP activity, which indicates NF-κB activation, was detected by using pNiFty2-SEAP plasmid and HEK-Blue^TM culture medium. The A₆₂₀ value of culture supernatant was detected by microplate a reader, and the intracellular NF-κB activation was also observed under a microscope. Our results showed that the CsHMGB1-159/127 proteins encoding 159 and 127 amino acids were expressed, and the relative molecular weights (M_r) were approximately 23 500 and 20 000, respectively. The A₆₂₀ values of supernatant from mouse macrophage stimulated with CsHMGB1-159 protein (1 and 10 μg/ml)for 24 hours were 0.66 ± 0.08 and 0.65 ± 0.03, respectively, which were higher than those in the control group (0.29 ± 0.02)(t = 11.1, 28.5; P < 0.01), and blue staining was observed in the cytoplasm. The A₆₂₀ values after 48 h reached 1.02 ± 0.08 and 1.07 ± 0.08, respectively, which were higher than those in the control group (0.62 ± 0.035)(t = 11.2, 12.9; P < 0.01), and the blue staining in the cytoplasm was stronger. Similarly, the A₆₂₀ values after 24 h stimulation with CsHMGB1-127 protein (1 and 10 μg/ml) were 0.52 ± 0.08 and 0.56 ± 0.08, respectively, which were higher than those in the control group(t =7.39, 8.37; P < 0.01), and the blue staining was observed in the cytoplasm. The A₆₂₀ values after 48 h reached 1.10 ± 0.05 and 0.90 ± 0.10, respectively, which were also higher than those in the control group (t = 20.30, 6.26; P < 0.01), and stronger blue staining was also observed in the cytoplasm. The A₆₂₀ values of CsHMGB1-159 protein (1 and 10 μg/ml) after 24 hours of stimulation were significantly higher than that of CsHMGB1-127 protein (t = 2.98, 2.75; P < 0.05). There were also significant differences in A₆₂₀ value after 48 h stimulation (t = 3.40, P < 0.01). The HMGB1 homologous proteins of Clonorchis sinensis encoding 159 and 127 amino acids can stimulate mouse macrophage NF-κB activation in vitro. The CsHMGB1-159 has stronger stimulatory effect than CsHMGB1-127 protein.