Cell senescence is an important anti-cancer mechanism and may contribute to cancer therapeutic outcome. The present study observed the effects of Tenglong Buzhong Decoction (TLBZD), a Chinese herbal formula, on senescence in human colon carcinoma LS-174-T cells. LS-174-T cells were treated with TLBZD, and morphology change was observed under a microscope. Cell senescence was identified by senescence-associated β-galactosidase (SA-β-gal) staining, and cell cycle was analyzed by flow cytometry. Expressions of p53, p21(WAF1/CIP1), p16 and RB and RB phosphorylation were detected by Western blot. After being treated with TLBZD, LS-174-T cells became enlarged and flattened by morphology; SA-β-gal staining was positive and cell cycle was arrested in G₀/G₁. In addition, up-regulations of p21(WAF1/CIP1) and p16, as well as inhibition of RB phosphorylation were detected in response to TLBZD treatment. Expressions of p53 and RB were unchanged after TLBZD treatment. TLBZD is effective in inducing cell senescence in human colon carcinoma LS-174-T cells, which may relate to up-regulations of p21(WAF1/CIP1) and p16 and inhibition of RB phosphorylation.