It is common to find the co-occurrence of aflatoxin B₁ (AFB₁), ochratoxin A (OTA), zearalenone (ZEA) in feed. These mycotoxins can be metabolized or transferred to raw milk in the forms of Aflatoxin M1 (AFM₁), ochratoxin A (OTA), ZEA, and α-zearalenol (α-ZOL), which are stable in processing of dairy products. The aim of the present study is to investigate the cytotoxicity of combined mycotoxins of AFM1, OTA, ZEA, and α-ZOL on the human colon adenocarcinoma cell lines (Caco-2) by using the tetrazolium salt (MTT) assay and the isobologram analysis. Statistical analysis of data was carried out using SAS9.2 statistical software package. Our results demonstrated the significant cytotoxic effects (P < 0.05) of the 2-toxin, 3-toxin and 4-toxin combination on Caco-2 cells in a time- and concentration-dependent manner. The results from MTT assay demonstrated that AFM₁ had the similar cytotoxicity as OTA (IC₅₀ = 4.10 μM) upon exposure for 72 h, which was much higher than that of ZEA and α-ZOL. The cytotoxic effect of the all the mycotoxin combinations increased in the following order: OTA+α-ZOL < ZEA+α-ZOL < OTA+ZEA < AFM₁+ZEA < AFM₁+α-ZOL < AFM₁+OTA < OTA+ZEA+α-ZOL < AFM₁+ZEA+OTA < AFM₁+ZEA+α-ZOL < AFM₁+OTA+α-ZOL < AFM₁+ZEA+OTA+α-ZOL. Isobologram analysis was further indicated that the existence of AFM1 brought additive effect at IC₅₀ and even synergism at IC₂₅. The synergistic effect of the 4-toxin group is the highest of all mytotoxin combinations investigated (Combination Index = 0.17 ± 0.05 at IC₂₅). Other cell lines will be further studied in the future to obtain an appropriate health risk assessment of milk.