Objective The cytotoxic effects of a new formulation of Pingyangmycin-activated carbon nanoparticles(PYM-CH-NP) on two human oral squamous cell carcinoma Tca8113 and BcaCD885 cell lines were studied in vitro. Methods The inhibitory effects of PYM-CH-NP and Pingyangmycin(PYM) were evaluated by methyl thiazolyl tetrazolium(MTT) assay at 1 -7 days. The 50% inhibition concentration values(IC50) and relative anti tumor activity(RAA) of PYM-CH-NP and PYM against Tca8113 and BcaCD885 with different drug concentration were evaluated. The time-dependent cytotoxic effects of PYM-CH-NP and PYM were during 1-5 days, so the doseeffect relationship was investigated at 5th day. Results Both PYM-CH-NP and PYM had high anticancer effects on Tca8113 and BcaCD885, and the cytotoxic effects were dose -dependent and time -dependent. Conclusion The activated carbon nanoparticles(CH-NP) may serve as a new drug delivery carrier of PYM. The new formulation PYM-CH-NP could slow down drug release, prolonged the drug concentration and its acting time, so more effective anticancer efficacy could be achieved.