This study aimed to evaluate the rumen protective effectiveness of L-carnitine through in vitro tests, rumen degradation tests and in vivo tests. Twelve rumen-fistulated crossbred rams with similar body weights of 55±3.6 kg and ages of 3±0.2 years old were divided into three treatment groups in a 3×3 Latin square design, G1 (basal diet with no additives), G2 (unprotected L-carnitine) or G3 (rumen-protected L-carnitine). Ruminal fluid and blood samples were collected before morning feeding on the last day of each experimental period (21 d). The percentage of L-carnitine remaining in the simulated rumen and abomasum and rumen increased with the increase in the wall material ratio (P< 0.05). L-carnitine supplementation decreased the plasma urea nitrogen concentration of the sheep (P< 0.05). G3 resulted in higher GSH-Px and SOD activities as well as T-AOC and lower MDA concentrations in plasma than G1, and the difference was significant among the groups (P< 0.01). Thus, L-carnitine in the rumen could be protected by encapsulation for a certain time. Unprotected and rumen-protected L-carnitine supplementation effectively enhanced the antioxidant capacity of sheep, and the antioxidant capacity of sheep supplemented with rumen-protected L-carnitine was higher than that of sheep supplemented with unprotected L-carnitine.