Objeetive： To study the in vitro anti-tumor effect of methotrexate （MTX） and vincristine （VCR）-loaded erythrocytes. Methods： MTX-4-VCR-loaded, MTX-loaded, and VCR-loaded erythrocytes were prepared by modified hypoosmotic swelling technique. K562 cells were treated with the prepared erythrocytes, pure erythrocytes, MTX＋VCR solution, and RPMI 1640 medium. MTT assay was used to examine the proliferation of K562 cells. The cell cycle of K562 was observed by flow cytometer using PI dyeing. The apoptosis of co-cultured K562 cells was analyzed by flow cytometer with Annexin VFITC/PI kit. Results： MTX ＋ VCR-loaded erythrocytes efficiently inhibited the proliferation and induced apoptosis of co-cultured K562 cells in a time-dependent manner, and its effects were better than those mono-loaded ones （P〈0. 05） and unloaded ones（P〈0.01）. Cellular cycle analysis demonstrated that, compared with control groups, proportion of K562 cells at G0/G1 phase decreased whereas that at S phase increased 12 hours after co-cultured with MTX＋VCR-loaded erythrocytes. 24 hours after co-cultured with MTX ＋ VCR-loaded erythrocytes the cells at G2/M phase increased and the cells in S phase decreased compared with those of 12 hours; there were significant difference between MTX＋VCR group and unloaded group; and the MTX＋VCR-loaded erythrocytes had a similar effect to that of MTX＋VCR solution. Conclusion： Compared with pure erythrocytes and mono-loaded erythrocytes, MTXA-VCR-loaded erythrocytes have more potent anti-tumor effect.