The excessive intake of trans fatty acids (TFAs) increases serum LDL-cholesterol and reduces HDL-cholesterol. Limited data exist regarding the low-level intake of TFAs, and the tolerable upper-limit level remains to be fully elucidated. A randomized, double-blind, parallel trial was conducted to assess the effects of a low level of TFA supplementation on serum cholesterol levels in healthy adult Japanese women. The volunteers who participated in this examination took in approximately 0.4% of energy (%E) TFAs from daily meals. Fifty-one volunteers consumed one cookie containing 0.6%E (TFA) or 0.04%E (control) of TFAs every day for 4 wk, and blood was harvested after overnight fasting. The mean TFA intakes of the control and TFA groups during the experimental period were 0.4%E and 1.1%E, respectively. There were no significant differences in serum total, LDL- or HDL-cholesterol levels between the control and TFA groups. The serum glucose and insulin levels were not influenced by TFA supplementation. These results confirm that dietary supplementation with 0.6%E TFAs (a total TFA intake of approximately 1%E) would have little effect on serum cholesterol levels in healthy adult Japanese women.