Senescent stromal cells support the development of prostate cancer and are considered potential therapeutic targets. This research evaluated the regulatory effects of ginsenoside Rg3 on the senescence of prostatic stromal cells pre-incubated in medium supplemented with 0.5% fetal bovine serum. The results revealed that ginsenoside Rg3 decreased the number of stromal cells positively stained with a senescent cell marker (senescence-associated β-galactosidase). Ginsenoside Rg3 also increased the viability of stromal cells and promoted cell cycle transition from G0/G1 to S phase, as well as inhibited the carcinoma-associated fibroblast-like phenotype in prostate stromal cells, through the up-regulation of smooth muscle cell markers SM22 and smooth muscle myosin heavy chain. Conditioned medium collected from stromal cells treated with ginsenoside Rg3 exhibited an attenuated effect on the promotion of prostate cancer cell migration compared with conditioned medium from stromal cells without Rg3 treatment. Down-regulation of interleukin 8 (IL-8) in a dose- and time-dependent manner was observed in ginsenoside Rg3-treated stromal cells, and over-expression or addition of IL-8 reversed the anti-senescence role of Rg3 in prostate stromal cells. Furthermore, ginsenoside Rg3 down-regulated IL-8 expression by decreasing the reactive oxygen species level in prostatic stromal cells and reducing the transcriptional activity of IL-8 promoter by damping the transcription factors C/EBP β and p65 binding to IL-8 promoter. Our research revealed that ginsenoside Rg3 was able to inhibit prostate stromal cell senescence by down-regulating IL-8 expression. The results suggest a potential value for ginsenoside Rg3 in prostate cancer treatment through the targeting of pro-carcinogenic senescent stromal cells.