The effects of the absence of protozoa in the rumen of lambs on animal growth, rumen fermentation, microbial diversity and fatty acid profiles in abomasal fluid and intramuscular fat were measured in ten control and ten protozoa-free (PF) lambs. PF lambs were prepared by isolating them from ewes within the first 24 h after birth. The PF and control lambs were kept for 4 months in two different fields and received a daily supplement of 250 g concentrate. The bacterial populations visualised by denaturing gradient gel electrophoresis differed between the two groups of animals and showed a higher bacterial diversity in control lambs than in PF lambs. Abomasal contents from control lambs contained higher concentrations of 22:5n-3 and 22:6n-3 and lower concentrations of vaccenic acid (trans-11-18: 1) and 20:3n-6 than PF lambs. The rest of the fatty acids, including the conjugated linoleic acid (CLA) isomers, were present at the same concentrations in abomasal contents from both experimental groups. Fatty acid composition in intramuscular fat showed differences between the groups. PF lambs had higher proportions of 18:0, 18:3, trans-10, cis-12-CLA and total SFA than control lambs. Control lambs had higher proportions of cis-9-18: 1, cis-9, cis-12-18:2, 20: 3n-6, 22:6n-3 (DHA) and MUFA. In conclusion, rumen defaunation led to higher tissue levels of the trans-10, cis-12-CLA isomer and SFA and lower PUFA:SFA ratio and n-3 PUFA in lamb muscle.