Abstract: Objective To explore the effect of interfering tumor microenvironment with low-dose cyclophosphamide (CYC) administration on homing and killing activity of adoptive transfused cytotoxic T lymphocytes in adoptive immunotherapy. Methods Melanoma-bearing mice models were established and randomly divided into four groups: control group, CYC injection group (CYCI), the solution of 0.9% w/v of NaCl injection plus CTL infusion group (NSI+CTL) and CYC injection plus CTL infusion group (CYCI+CTL), 40 cases in each group. CYC (20 mg/kg) were intraperitoneally injected in mice of CYCI group and CYCI+CTL group. The same amount of the solution of 0.9% w/v of NaCl was injected in mice of NSI+CTL group. The levels of Treg, TGF-β and IL-10 were detected at the different time points before and after CYC or the solution of 0.9% w/v of NaCl injection. The cultured CFSE-CTLs were transfused at the 4th day after injection, 5×l0⁶ cells per mouse. Then, CFSE-CTLs ratio was analyzed by FCM. Results In NSI+CTL group, the levels of Treg, TGF-β and IL-10 were all increased continuously with tumor growth. CFSE-CTLs ratio in CYCI+CTL group was significantly higher and lasted longer than that in NSI+CTL group (P<0.05). There was significant difference among every two group, except control group and CYCI group(P<0.05). Conclusion Tumor microenvironment in tumor-bearing mice could be effectively intervened by low-dose CYC administration in advance, with decreased Tregs, IL-10, and TGF-β levels. As a result, more transferred CTLs homing leads to the strenthened killing efficacy.