Background: We sought to elucidate the effects of levobupivacaine on modulating endotoxin-induced upregulation of inflammatory mediators and activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways in activated microglia. Materials and methods: Confluent murine microglia (BV-2) were treated with endotoxin (lipopolysaccharide, 50 ng/mL) or endotoxin plus levobupivacaine (5, 25, or 50 μM) and denoted as the LPS, LPS + L(5), LPS + L(25), and LPS + L(50) groups, respectively. Levobupivacaine was administered immediately after endotoxin. Control groups were run simultaneously. Results: The concentrations of inflammatory mediators, including macrophage inflammatory protein-2 (P = 0.023 and 0.016), tumor necrosis factor-α (P = 0.025 and 0.020), interleukin (IL)-1β (P = 0.018 and 0.014), IL-6 (P = 0.029 and 0.023), nitric oxide (P = 0.025 and 0.026), and prostaglandin E2 (P = 0.028 and 0.016) of the LPS + L(25) and LPS + L(50) groups were significantly lower than those of the LPS group. The concentrations of macrophage inflammatory protein-2 (P = 0.035), IL-1β (P = 0.024), nitric oxide (P = 0.031), and prostaglandin E 2 (P = 0.036) but not tumor necrosis factor-α and interleukin-6 of the LPS + L(5) group were also significantly lower than those of the LPS group. These data revealed that effects of endotoxin on upregulating inflammatory mediators were inhibited by levobupivacaine. Moreover, effects of endotoxin on activating NF-κB, including inhibitor-κB degradation, NF-κB nuclear translocation, and NF-κB-DNA binding, were also inhibited by levobupivacaine. Similarly, effects of endotoxin on activating MAPKs, including extracellular signal-regulated kinase, c-jun N-terminal kinase, and p38 MAPK, were also significantly inhibited by levobupivacaine. Conclusions: Levobupivacaine significantly inhibited endotoxin-induced upregulation of inflammatory mediators and activation of NF-κB and MAPKs signaling pathways in activated microglia.