Background: Trans fatty acids are produced either by industrial hydrogenation or by biohydrogenation in the rumens of cows and sheep. Industrial trans fatty acids lower high-density lipoprotein (HDL) cholesterol, raise low-density lipoprotein (LDL) cholesterol, and increase the risk of coronary heart disease. The effects of trans fatty acids from ruminants are less clear. We investigated the effect on blood lipids of cis-9, trans-11 conjugated linoleic acid (CLA), a trans fatty acid largely restricted to ruminant fats. Methodology/Principal Findings: Sixty-one healthy women and men were sequentially fed each of three diets for three weeks, in random order, for a total of nine weeks. Diets were identical except for 7 % of energy (approximately 20 g/day), which was provided either by oleic acid, by industrial trans fatty acids, or by a mixture of 80 % cis-9, trans-11 and 20 % trans-10, cis-12 CLA. After the oleic acid diet, mean (6 SD) serum LDL cholesterol was 2.6860.62 mmol/L compared to 3.0060.66 mmol/L after industrial trans fatty acids (p,0.001), and 2.9260.70 mmol/L after CLA (p,0.001). Compared to oleic acid, HDL-cholesterol was 0.0560.12 mmol/L lower after industrial trans fatty acids (p = 0.001) and 0.0660.10 mmol/L lower after CLA (p,0.001). The total-to–HDL cholesterol ratio was 11.6 % higher after industrial trans fatty acids (p,0.001) and 10.0 % higher after CLA (p,0.001) relative to the oleic acid diet. Conclusions/Significance: High intakes of an 80:20 mixture of cis-9, trans-11 and trans-10, cis-12 CLA raise the total to HDL