The aim of this study was to investigate the effects of dietary conjugated linoleic acid (CLA) on the cytotoxicity of peripheral blood lymphocytes in piglets. A total of forty 21-d-old Duroc × Large White × Landrace weaned piglets were randomly allocated into two treatment groups and fed with control diet or 2% CLA diet for 6 weeks. Dietary CLA significantly increased the number of peripheral blood CD8 T lymphocytes (P < 0.05). Compared with the control group, cytotoxicity, perforin, and granzyme B mRNA expression of peripheral blood lymphocytes in piglets from the CLA-supplementation group increased by 29.98% (P < 0.01), 32.87% (P < 0.01), and 43.36% (P < 0.05), respectively. The piglets on the CLA diet had higher mRNA expression of Lck and Fyn in peripheral blood lymphocytes (P < 0.05). The content of C16:0 significantly increased in piglets from the dietary CLA-supplementation group (P < 0.05), whereas the content of C18:1 significantly decreased (P < 0.05). The CLA content of peripheral blood lymphocytes in piglets in the CLA diet group ranged up to 0.24% (P < 0.01). These results suggest that dietary CLA enhanced cytotoxicity of peripheral blood lymphocytes perhaps through changing its fatty acid composition.