This study was performed to characterize the CLA production ability of a bacterial strain, Bifidobacterium breve LMC 520, which can actively convert linoleic acid (LA) to cis-9,trans-11 conjugated linoleic acid (CLA), a major isomer derived from microbial enzymatic conversion. The culture conditions were optimized to improve CLA production under the aerobic conditions. B. breve LMC 520 was tested with different amounts of LA in varied culture conditions, such as air, additives, and pH. A maximal level of CLA production (up to 90% of substrate) was obtained after 24 h of incubation in culture medium containing 1 mM LA at pH 5.5 and under anaerobic conditions. There was no decline in the CLA level with prolonged incubation until 48 h. When the effect of pre-incubation with LA on CLA production was tested, there was no significant difference between the CLA-producing activity of pre-incubated and untreated bacteria at the third passage but there was a significant reduction in CLA production by the pre-incubated cells after the fourth passage. These results demonstrate that the CLA-producing activity of B. breve LMC 520 could be maximized by numerous environmental factors. The data also indicate its potential for increasing CLA accumulation in dairy products when B. breve LMC 520 is used as a functional starter culture.