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Rotifer are important food resources in aquaculture hatcheries, and several studies have shown that L-carnitine (LC) can promote the growth performance of rotifers. The aim of this study was to investigate the nature of beneficial effects of LC on rotifer Brachionus rotundiformis. The feeding behavior of rotifers were evaluated at different LC concentrations (0, 1, 10 and 100 mg L−1). Meanwhile, the effects of LC on growth performance, free LC content, amino acid and fatty acid composition of rotifers were analyzed. The results indicated that LC (10 and 100 mg L−1) treatments significantly improved the filtration and ingestion rates of rotifers fed microalgae or baker’s yeast, In addition, LC (10 and 100 mg L−1) not only significantly accumulated in rotifers, but also significantly improved the rotifer growth performance. After 48 h of LC treatment, the rotifer population density significantly increased. Concerning the biochemical composition of rotifers, 100 mg L−1 LC can increase the carbohydrates content, decrease the protein and lipid content in the rotifers. And the LC treatment could significantly decrease the total SFA content of rotifers, while significantly increase the total HUFA content of rotifers. Moreover, the levels of LC supplementation were positively correlated with the contents of Thr, Gly, Ala, Leu, Tyr, Phe, Lys, His, Arg and Pro in rotifers. Overall, adding LC could not only accumulate in rotifers, but also promote the growth performance of rotifers by improving their feeding behavior and their nutritional value.