Introduction: Stem cells isolated from the root apical papilla of human teeth (stem cells from the apical papilla [SCAPs])are capable of forming tooth root dentin and are a feasible source for bioengineered tooth root regeneration. In this study, we examined the effect of acetylsalicylic acid (ASA)on odontogenic differentiation of SCAPs in vitro and in vivo. Methods: SCAPs were cultured under odontogenic conditions supplemented with or without ASA. ASA-treated SCAPs were also subcutaneously transplanted into immunocompromised mice. Results: ASA accelerates in vitro and in vivo odontogenic differentiation of SCAPs associated with down-regulation of runt-related nuclear factor 2 and up-regulation of specificity protein 7, nuclear factor I C, and dentin phosphoprotein. ASA up-regulated the phosphorylation of AKT in the odontogenic SCAPs. Of interest, pretreatments with phosphoinositide 3-kinase inhibitor LY294402 and small interfering RNA for AKT promoted ASA-induced in vitro and in vivo odontogenic differentiation of SCAPs. LY294402 and small interfering RNA for AKT also suppressed the ASA-induced expression of runt-related nuclear factor 2 and enhanced ASA-induced expression of specificity protein 7, nuclear factor I C, and dentin phosphoprotein in SCAPs. Conclusions: These findings suggest that a combination of ASA treatment and suppressive regulation of the phosphoinositide 3-kinase–AKT signaling pathway is a novel approach for SCAP-based tooth root regeneration.