In this study, the effects of high Trans Fatty Acid (TFA) intake on fatty acid contents in erythrocyte membranes were examined. Thirty-two rabbits were randomly divided into 4 groups: a control group fed a common diet, a high TFA group fed a normal diet supplemented with 5.0 g/kg/d TFA, a High-Fat (HF) group fed a high cholesterol diet, and a TFA+HF group. Saturated FAs, unsaturated FAs and TFA contents of erythrocyte membranes were determined at 0, 4, 8 and 12 weeks. We found that the TFA and saturated FA contents in erythrocyte membranes were significantly higher in the TFA group than in the control group, but ω-3 FAs were lower after 12 weeks. The ω-3/ΣTFA and ω-3/C18:0 ratios were also significantly lower in the TFA group than the control group. The ratios were similar in the HF group and the TFA group. In the TFA+HF group, the abnormalities in the FA ratios in the erythrocyte membrane were more obvious. High TFA intake could decrease polyunsaturated fatty acids in the erythrocyte membrane, especially ω-3 fatty acids, and decrease the ratios of ω-3 to TFA and saturated fatty acids. The effects of high cholesterol intake were equivalent to the effects of high TFA intake. Synergistic effects were clearly observed after combined TFA and high cholesterol intake.