The effects of lead (Pb) on endogenous nitric oxide (NO) generation, the role of NO in Pb uptake and the origin of Pb-induced NO production in Pogonatherum crinitum root cells were evaluated. Pb treatment induced rapid NO generation, showing that Pb exposure triggered endogenous NO signaling of the cells. Pre-treatment of the cells with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline –1-oxyl-3-oxide (cPTIO) not only abolished the Pb-triggered NO burst but also reduced Pb contents of the cells. Moreover, Pb exposure enhanced nitrate reductase (NR) activity of the cells. The NR inhibitors tungstate and glutamine not only suppressed the Pb-enhanced NR activities but also reduced the Pb-triggered NO generation. Pre-treatment of the cells with tungstate and glutamine suppressed Pb accumulation and the suppression could be restored by application of exogenous NO via its donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Together, our results indicated that Pb exposure enhanced NR activity and triggered the NO burst of P. crinitum root cells. Furthermore, the data demonstrated that NR was responsible for the Pb-triggered NO burst and that NR-mediated NO generation played a critical role in Pb uptake by P. crinitum root cells. Thus, our results suggest a potential strategy for controlling Pb uptake by plants by targeting NR as a source of Pb-triggered NO production.