Regulation of neutrophil apoptosis plays a critical role in the inflammatory response. Inflammation has previously been shown to increase levels of extracellular β-nicotinamide adenine dinucleotide (NAD(+)). The present study demonstrates that extracellular NAD(+) at concentrations found in the inflamed tissues profoundly delays spontaneous apoptosis of human neutrophils as was evidenced by inhibition of phosphatidylserine (PS) exposure, DNA fragmentation and caspase-3 activation. The effect was abrogated by NF157, an antagonist of P2Y11 receptor, and was pertussis toxin-insensitive. The NAD(+)-mediated delay of neutrophil apoptosis was reversed by 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, and Rp-8-Br-cAMPS, an inhibitor of type I cAMP-dependent protein kinase A (PKA). Blocking of NAD(+)-induced influx of extracellular Ca(2+) with EGTA did not abolish the pro-survival effect of NAD(+). Extracellular NAD(+) inhibited proteasome-dependent degradation of Mcl-1 upstream of caspase activation and, furthermore, suppressed Bax translocation to the mitochondria and attenuated both dissipation of mitochondrial transmembrane potential (ΔΨm) and cytochrome c release from the mitochondria into the cytosol. Finally, we found that extracellular NAD(+) inhibited spontaneous activation of caspase-9, but not caspase-8, and the pro-survival effect of extracellular NAD(+) was abrogated by the inhibitor of caspase-9, but not by the inhibitor of caspase-8. Together, these results demonstrate that extracellular NAD(+) inhibits neutrophil apoptosis via P2Y11 receptor and cAMP/PKA pathway by regulating Mcl-1 level, Bax targeting to the mitochondria and mitochondrial apoptotic pathway. Thus, extracellular NAD(+) acts as a neutrophil survival factor that can contribute to prolonged neutrophil lifespan in inflammatory response.