Stem cell–based therapeutics is a promising strategy in dental pulp regeneration. However, low cell viability after transplantation in vivo due to the ischemic microenvironment is still a critical challenge for future clinical application. With the aim of improving postimplantation cell survival and pulp tissue regeneration, stem cells from human exfoliated deciduous teeth (SHED) were preconditioned to a hypoxic condition by hypoxia-inducible factor 1α (HIF-1α) stabilization via knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) using lentiviral short hairpin RNA. HIF-1α–stabilized SHED were encapsulated in PuraMatrix hydrogel, injected into root canals of human tooth fragments, and implanted in the subcutaneous space of immunodeficient mice. After 28 d, enhanced dental pulp–like tissue formation was observed with a significantly higher level of vascularization, which could be attributed to both endothelial differentiation of SHED and recruitment of host blood vessels. Furthermore, dentin-like tissue formation in vivo and accelerated odontogenic/osteogenic differentiation both in vivo and in vitro were observed. At 7 d postimplantation, significantly less DNA damage and higher Ki67 expression were detected in the HIF-1α–stabilized SHED group compared with the control SHED. Accordingly, cell viability assay and staining for Ki67 and apoptotic cells in vitro showed that HIF-1α stabilization could decrease cell apoptosis and enhance cell survival significantly. We demonstrated that PI3K/AKT pathway activation had resulted in low caspase 3 expression in HIF-1α–stabilized SHED in hypoxic conditions. Furthermore, we found that HIF-1α–induced cell survival could also be attributed to the upregulated expression of PDK1, HK2, and Glut1, which contributes to the maintenance of reactive oxygen species homeostasis and metabolic adaptation in hypoxia. In addition, we identified Smad7 as 1 of the top 3 upregulated genes through RNA sequencing in HIF-1α–stabilized SHED and demonstrated its essential role in HK2 and Glut1 upregulation. Taken together, HIF-1α stabilization enhances cell survival of SHED through modulating various target genes and potential signaling pathways, as well as odontogenic tissue formation during dental pulp regeneration, which could benefit stem cell–based therapy in general.