Introduction:Porphyromonas gingivalisandEnterococcus faecalispromote the development of pulpitis and periapical periodontitis. These bacteria are difficult to eliminate from the root canal systems, leading to persistent infection and poor treatment outcomes. We explored the response of human dental pulp stem cells (hDPSCs) to bacterial invasion and the mechanisms underlying the impact of residual bacteria on dental pulp regeneration.Methods:Single-cell sequencing was used to categorize the hDPSCs into clusters based on their response toP. gingivalisandE. faecalis. We depicted a single-cell transcriptome atlas of hDPSCs stimulated byP. gingivalisorE. faecalis.Results:The most differentially expressed genes in the Pg samples wereTHBS1,COL1A2,CRIM1, andSTC1, which are related to matrix formation and mineralization, andHILPDAandPLIN2, which are related to the cellular response to hypoxia. A cell cluster characterized by high expression levels ofTHBS1andPTGS2was increased afterP. gingivalisstimulation. Further signaling pathway analysis showed that hDPSCs preventedP. gingivalisinfection by regulating the TGF-β/SMAD, NF-κB, and MAPK/ERK signaling pathways. Differentiation potency and pseudotime trajectory analyses showed that hDPSCs infected byP. gingivalisundergo multidirectional differentiation, particularly to the mineralization-related cell lineage. Furthermore,P. gingivaliscan create a hypoxia environment to effect cell differentiation. The Ef samples were characterized by the expression ofCCL2, which is related to leukocyte chemotaxis, andACTA2, which is related to actin. There was an increased proportion of a cell cluster that was similar to myofibroblasts and exhibited significantACTA2expression. The presence ofE. faecalispromoted the differentiation of hDPSCs into fibroblast-like cells, which highlights the role of fibroblast-like cells and myofibroblasts in tissue repair.Discussion:hDPSCs do not maintain their stem cell status in the presence ofP. gingivalisandE. faecalis. They differentiate into mineralization-related cells in the presence ofP. gingivalisand into fibroblast-like cells in the presence ofE. faecalis. We identified the mechanism underlying the infection of hDPSCs byP. gingivalisandE. faecalis. Our results will improve understanding of the pathogenesis of pulpitis and periapical periodontitis. Furthermore, the presence of residual bacteria can have adverse effects on the outcomes of regenerative endodontic treatment.