The objective of the present study was to investigate the effects of a lipid-encapsulated CLA preparation on rumen metabolism and the actual post-ruminal bioavailability of the applied CLA isomers. In the rumen, the CLA supplementation modified the molar proportions of VFA. In period CLA-1 the rumen fermentation shifted toward more butyric acid at the expense of acetic acid. The highest CLA supplementation resulted in increased amounts of isobutyric, isovaleric, and valeric acid. The apparent ruminal digestibility of starch increased in period CLA-2. The ruminal protein degradation was higher after CLA supplementation, while the efficiency of the use of the RDP for microbial protein synthesis declined. The duodenal flow of trans-10,cis-12 CLA amounted to 16 and 5% of the intake in periods CLA-1 and CLA-2, respectively. The transfer of trans-10,cis-12 CLA from duodenum into milk was 36 and 48% in periods CLA-1 and CLA-2, respectively. Overall, the observed effects of the supplementation of lipid-encapsulated CLA on the parameters of rumen metabolism were negligible. The actual low post-ruminal bioavailability of trans-10,cis-12 CLA suggest that most of the applied fat supplement was biohydrogenated.