AbstractAcupuncture has been used for millennia to treat pain, although its efficacy and duration of action is limited. Acupuncture also has brief (1–2 h) antinociceptive effects in mice and these effects are dependent on localized adenosine A1 receptor (A1R) activation. Intriguingly, adenosine 5’-monophosphate (AMP) is basally elevated near acupuncture points. This finding suggested that it might be possible to inhibit nociception for a longer period of time by injecting prostatic acid phosphatase (PAP, ACPP) into acupuncture points. PAP is an ectonucleotidase that dephosphorylates extracellular AMP to adenosine, has a long half-life in vivo and is endogenously found in muscle tissue surrounding acupuncture points. Here, we found that injection of PAP into the popliteal fossa—a space behind the knee that encompasses the Weizhong acupuncture point—had dose- and A1R-dependent antinociceptive effects in mouse models of acute and chronic pain. These inhibitory effects lasted up to six days following a single injection, much longer than the hour-long inhibition provided by acupuncture. Antinociception could be transiently boosted with additional substrate (AMP) or transiently blocked with an A1R antagonist or an inhibitor of phospholipase C. This novel therapeutic approach—which we term “PAPupuncture”—locally inhibits pain for an extended period of time (100x acupuncture), exploits a molecular mechanism that is common to acupuncture, yet does not require acupuncture needle stimulation.