Tris (2-chloroethyl) phosphate (TCEP) has been widely used as a plasticizer and flame retardant. TCEP as a potential carcinogen is often detected in the occupational and nature environments. To investigate effects of TCEP on human hepatocytes, we assessed cell growth rate, cellular membrane integrity, senescence-associated β-galactosidase (SA-β-Gal) activity and analyzed expression of regulators involved in the p53-p21(Waf1/Cip1)-Rb pathway in TCEP-treated L02 cells. The results showed TCEP increased the percentage of SA-β-Gal positive cells, decreased IL-6 levels, down-regulated the regulators of p38MAPK-NF-κB pathways, but up-regulated the regulators of p21(Waf1/Cip1)-Rb pathway in L02 cells. Furthermore, we measured the SA-β-Gal activity and expression of regulators involved in the p53-p21(Waf1/Cip1)-Rb pathway in L02(-p53) cells and p53-null Hep3B cells. Similar results were found in L02(-p53) cells and Hep3B cells. The findings demonstrated that TCEP induced senescence-like growth arrest via the p21(Waf1/Cip1)-Rb pathway in a p53-independent manner, without activation of the IL-6/IL6R, p38MAPK-NF-κB pathways in hepatocytes.