Abstract: Objective: To study the protective effect and mechanism of ethanol extract from Paeonia suffruticosa seed on photoaging of HaCaT cells induced by UVB. Method: The anti-photoaging active ingredients in the ethanol extract of Paeonia suffruticosa seed were analyzed using ultra-performance liquid chromatography/quadrupole time-of-flight-tandem mass spectrometry (UPLC-Q-TOF-MS), and the photoaging cell model was established by stimulating HaCaT cells with UVB. Cell viability was determined by the multiple table tournament (MTT) method. The effect of ethanol extract from Paeonia suffruticosa seed on cell migration was detected by cell scratch assay. The related cytokines affecting senescence such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-22 (IL-22), interferon-γ (IFN-γ), and tansforming growth factor-β (TGF-β) were detected by enzyme-linked immunosorbent (ELISA) experiment, and the levels of reactive oxygen species (ROS) and nuclear factor-κ-gene binding (NF-κB) were used as indicators to study the anti-photoaging activity and mechanism of Paeonia suffruticosa seed ethanol extract at different concentrations on HaCaT cells. Results: The top five components with the highest content were 1-stearoylglycerol (10.73%), erucamide (3.17%), 5-(2R,3S)-6-hydroxy-2-(4-hydroxyphenyl)-4-(E)-2-(4-hydroxyphenyl)ethenyl-2,3-dihydro-1-benzofuran-3-ylbenzene-1,3-diol (2.20%), α,α-trehalose (1.90%) and albiflorin (1.45%). After treatment with different concentrations of Paeonia suffruticosa seed ethanol extract, the viability of HaCaT cells was above 80%. The concentration of 6.25 μg/mL of Paeonia suffruticosa seed ethanol extract showed no significant cytotoxicity to HaCaT cells. Both the concentrations of 12.5 μg/mL (H group) and 6.25 μg/mL (L group) of Paeonia suffruticosa seed ethanol extract could inhibit the migration ability of HaCaT cells. The H group significantly reduced the levels of IL-1, IL-6, IL-22, IFN-γ, and TGF-β in cells (P<0.05), thereby resisting skin aging. The L group reduced ROS production and decreased the content of NF-κB protein factor (P<0.05), effectively inhibiting the photoaging response of HaCaT cells. Conclusion: Ethanol extract from Paeonia suffruticosa seed can improve oxidative stress and inflammation induced by UVB, and has the effect of anti-photoaging.