The present study evaluates the effect of dietary trans fatty acids on diaphragm phospholipid fatty acid composition, intramyocellular triacylglycerol content and insulin-stimulated glucose uptake in comparison with dietary saturated fatty acids. Male weanling WNIN rats were divided into three groups and fed for 3 months on one of the following diets containing 10 % oil differing in fatty acid composition: control diet, saturated fatty acid diet and trans fatty acid diet. Dietary trans fatty acids increased the intramyocellular triacylglycerols and decreased the ratio of 20 : 4n-6 to 18 : 2n-6 and long-chain PUFA levels (20 %) in diaphragm phospholipids, indicating inhibition of PUFA biosynthesis. However, saturated fatty acids decreased both 18 : 2n-6 and 20 : 4n-6 without change in the ratio. Trans fatty acid-induced alterations in diaphragm phospholipid fatty acid composition and intramyocellular triacylglycerol content were associated with decreased insulin-stimulated glucose transport in the diaphragm. These observations suggest that dietary trans fatty acids decrease diaphragm insulin sensitivity, possibly due to increased intramyocellular triacylglycerol accumulation and decreased long-chain PUFA in phospholipids.