The present study was conducted to examine the effects of different plant oils or plant oil mixtures and high-temperature, microtime processing (HTMT) on the CLA content in Hanwoo steers. Experiment 1, consisting of 3 in vitro trials, was conducted to determine how the biohydrogenation of C18 fatty acids and CLA production were affected by fat sources (tallow, soybean oil, linseed oil, or mixtures of soybean oil and linseed oil) or HTMT treatment in the rumen fluid. The results showed that HTMT was capable of protecting unsaturated fatty acids from biohydrogenation by ruminal bacteria. The HTMT-treated diet containing 4% linseed oil (LU) and a supplement containing 2% linseed oil and 1% soybean oil treated with HTMT + 1% soybean oil (L(2)S(1)U+S(1)) produced an increased quantity of trans-11 C18:1 and cis-9, trans-11 CLA, and a reduced quantity of trans-10, cis-12 CLA. Based on these results, in vivo studies (Exp. 2) were conducted with LU and L(2)S(1)U+S(1). These 2 treatments increased the content of cis-9, trans-11 CLA in LM compared with the control diet. The content of trans-10, cis-12 CLA in subcutaneous fat was also increased in the L(2)S(1)U+S(1) treatment compared with other treatments. The subcutaneous fat thickness in the LU treatment was decreased compared with the L(2)S(1)U+S(1) treatment. The LU treatment significantly decreased fatty acid synthase expression but simultaneously increased leptin expression. In this report, we showed that diets containing LU and L(2)S(1)U+S(1) were capable of increasing CLA in the intramuscular fat of beef.