This experiment studied the effect of a modest difference in diet structure value (SV) on milk conjugated linoleic acid (CLA) contents of cows fed diets supplemented with extruded linseed, in situations where the diets provided enough SV and therefore did not induce milk fat depression. Six lactating Holstein cows were used in a crossover design with two treatments (‘SV 1.50’ and ‘SV 1.73’) and two periods of 21 days. The ‘SV 1.50’ diet contained 59% maize silage, 13% soya bean meal, 13% sugar beet pulp and 14% Nutex Compact (containing 56% extruded linseed) (dry matter (DM) basis) and was offered as a restricted total mixed ration. For the ‘SV 1.73’ diet, 8% wheat straw (DM basis) was added to the ‘SV 1.50’ diet as an additional structure source. The two diets had a forage-to-concentrate ratio of 59 : 41 and 62 : 38. The inclusion of straw in the diet resulted in an additional intake of NDF (+1110 g/day), which accounted for 90% of the additional intake of OM, whereas additional intakes of the other nutrients were minor. Milk yield and composition did not differ among treatments. The inclusion of straw in the diet did not affect the milk levels of t10–18:1, 18:2n-6, c9-16:1, c9-18:1, c11-18:1, 6:0, 8:0, 20:4 and 20:5. It decreased the milk levels of c9,t11-CLA (2.13% v. 3.03% of fatty acids (FA) reported, P < 0.001), t11-18:1 (4.99% v. 7.10% of FA reported, P < 0.001), 18:3n-3, t9-16:1 and t9-18:1, while it increased the milk levels of 6:0–14:0 (20.90% v. 19.69% of FA reported, P < 0.01), 16:0 (26.55% v. 25.25% of FA reported, P < 0.01), 18:0 (13.54% v. 12.59% of FA reported, P < 0.001), 17:0, 20:0 and 22:5. Regarding the ratio between FA, the inclusion of straw increased the 18:0/total C18 FA ratio (37.74% v. 32.07%, P < 0.001), whereas it decreased the total trans-C18 FA/total C18 FA ratio (15.46% v. 20.34%, P < 0.001), the t11-18:1/total C18 FA ratio (13.70% v. 17.95%, P < 0.01) and the c9,t11-CLA/total C18 FA ratio (5.82% v. 7.64%, P < 0.001). We conclude from this experiment that even a modest increase in SV to a diet supplemented with extruded linseed, yet already providing enough SV, alters the rumen lipid metabolism and, hence, CLA levels in milk fat.