〔Abstract〕 Objective To investigate the role of runt-related transcription factor 3 (RUNX3) in transforming growth factor-β 1 (TGF-β 1)–induced activation of mouse primary pulmonary fibroblasts (PFs), and its effects on fibroblast activation protein (FAP) expression, cell proliferation, and collagen synthesis. Methods PFs were isolated from C57BL/6 mice and cultured. A RUNX3 knockdown model was established using small interfering RNA (siRNA). Cells were assigned to the control group (Control), TGF-β 1-treated group (TGF-β 1), negative control group (TGF-β 1+siRNA-NC), and RUNX3-silenced group (TGF-β 1+si-RUNX3). In addition, a RUNX3 overexpression rescue experiment was performed based on TGF-β 1stimulation. Protein and mRNA levels of RUNX3, FAP, and typeⅠcollagen (COL1A1) were measured by Western blot and reverse transcription quantitative real-time PCR (RT-qPCR). Cell proliferation was assessed using CCK-8 and EdU assays. Co-expression of COL1A1 and FAP was examined by double immunofluorescence staining. Results Compared with the Control group, RUNX3, FAP, and COL1A1 expression levels were upregulated in PFs in the TGF-β 1 group ( P < 0.01). The CCK-8 assay showed that the absorbance value was reduced in the RUNX3 knockdown group compared with the negative control group ( P < 0.01). Consistently, the EdU assay demonstrated a lower proportion of EdU-positive cells in the RUNX3 knockdown group than in the negative control group ( P < 0.01). Immunofluorescence double staining revealed decreased fluorescence intensities of COL1A1 and FAP in the RUNX3 knockdown group relative to the negative control. Under RUNX3 overexpression conditions, these fluorescence signals exhibited a partial rebound ( P < 0.01). ConclusionRUNX3 in TGF-β1–induced PFs may promote cell proliferation and collagen synthesis by positively regulating FAP expression. Targeting the RUNX3/FAP axis may represent a potential therapeutic strategy for pulmonary fibrosis.