<Introduction> High mobility group box 1 (HMGB1), a nuclear protein, is passively released by dead or dying cells, and actively secreted by certain alive cells such as activated macrophages (Mφ), contributing to various biological events such as inflammation, proliferation, and differentiation. Extracellular HMGB1 targets multiple molecules including Toll-like receptor 4 (TLR4), the receptor for advanced glycation end products (RAGE), CXC chemokine receptor 4 (CXCR4), and NMDA receptors [1]. It has been reported that HMGB1 contributes to neuropathic pain after peripheral nerve injury [2] and to axonal regeneration after spinal cord injury [3]. Given the activation of resident and infiltrating Mφ around injured peripheral neurons, we hypothesized that HMGB1 secreted by Mφ plays a role in the repair of injured neurons. We thus investigated the impact of exogenous and endogenous HMGB1 on the neuritogenesis of neuron-like NG108-15 cells in the presence and absence of Mφ-like RAW264.7 cells. <Methods> Extent of neuritogenesis in NG108-15 cells is shown as the proportion (%) of cells with longer neurites than the cell body diameter. In a co-culture assay, using 24-well plates with transwell inserts, NG108-15 and RAW264.7 cells were seeded in the plate wells and transwell inserts, respectively. Protein expression of TLR4 and secreted HMGB1 were determined by Western blotting. Data are shown as mean ± SEM. Statistical significance was evaluated by Student’s t-test or ANOVA followed by Tukey’s test. <Results> In NG108-15 cells, stimulation with HMGB1 at 0.5 mg/ml for 24 hours increased neuritogenesis, which was completely inhibited by MK-801, an NMDA receptor antagonist, at 10 µM [vehicle (V)+V 24.4±3.4%, V+HMGB1 50.4±4.5% (p<0.01 vs. V+V), MK-801+HMGB1 5.8±2.3% (p<0.01 vs. V+HMGB1), n=4]. Lipopolysaccharide (LPS) at 1 µg/ml, a TLR4 agonist, did not cause neuritogenesis (V 18.4±2.3%, LPS 24.9±2.5%, n.s., n=6), and TLR4 protein was not detected in NG108-15 cells. In RAW264.7 cells, LPS stimulated secretion of HMGB1 [V 0.007±0.005, LPS 0.337±0.050 (arbitrary unit), p<0.01, n=4]. In NG108-15 cells co-cultured with RAW264.7 cells, stimulation with LPS at 1 µg/ml for 72 hours caused neuritogenesis, an effect eliminated by an anti-HMGB1-neutralizing antibody (HMGB1-Ab) at 10 µg/ml or MK-801 at 10 µM [V+V 14.5±1.8%, V+LPS 31.6±3.9% (p<0.05 vs. V+V), control IgY+LPS 33.9±2.5% (n.s. vs. V+LPS), HMGB1-Ab+LPS 16.3±3.5% (p<0.01 vs. V+LPS); V+V 20.3±1.9%, V+LPS 33.5±2.1% (p<0.05 vs. V+V), MK-801+LPS 19.2±1.2% (p<0.01 vs. V+LPS), n=4-6]. <Conclusions> Our data thus suggest that Mφ-derived HMGB1 promotes neuritogenesis via NMDA receptors in the neuron-like NG108-15 cells.