Ｏｂｊｅｃｔｉｖｅ　Investigating whether bupivacaine induces decline of intracellular nicotinamide adenine dinucleotide (NAD) level, and whether NAD level decreasing is involved in bupivacaine-induced neurotoxicity.　Methods　SH-SY5Y cell were treated with 1 mmol/L for indicated time points (30 min to 7 h), the intracellular NAD content and cell viability were detected. SH-SY5Y cells were treated with bupivacaine at concentrations varying from 1 mmol/L to 10 mmol/L for 30 min. The intracellular NAD content and cell viability were then detected. SH-SY5Y cells exposed to 5 mmol/L bupivacaine for 30 min, upon 30 min pre-, post- NAD treatment at different concentrations. The intracellular NAD levels and the cell viabilities in all groups were examined.　Results　The intracellular NAD level began to decline after 3 h bupivacaine treatment. The level was decreased to (27.8±8.47)% at 7 h time point of treatment. The intracellular NAD content of SH-SY5Y cells were decreased to (21.50±3.15)%, (25.73±7.22)%, and (16.07±13.93)% respectively after receiving 2, 5, 10 mmol/L bupivacaine treatment for 30 min. Thus, the content levels of NAD were significantly lower than untreated control(P＜0.05). Upon same conditions, there were no significant differences among different experimental groups with 1 mmol/L bupivacaine treatment at indicated time points(P＞0.05). And cell viability were also decreased while concentrations of bupivacaine increasing[cell viability significantly declined to (49.44±8.55)%, (35.75±15.83)%, and (25.58±4.45)%, respectively, at the concentration of 2, 5, 10 mmol/L (P＜0.05). The cellular NAD levels reached (85.87±11.82)%,(89.21±11.55)%, and (105.05±58.82)% in 2.5, 5, 10 mmol/L NAD pre-treatment groups respectively. The cellular NAD levels were significantly higher than the levels in bupivacaine groups(P＜0.05). The cell viabilities were higher in cells receiving NAD pre-treatment or post-treatment than viability of cells were treated with 5 mmol/L bupivacaine alone. Thus, the pre-treatment group achieved better viability(P＜0.05).　Conclusions　Intracellular NAD level decline was involved in bupivacaine induced neurotoxicity. Exogenous NAD increases cellular NAD level and attenuates the cytotoxicity resulting from bupivacaine.