L-proline (L-PROL) is an essential amino acid, a constituent of many proteins and the osmoprotective molecule produced and accumulated in higher plants and some freshwater microalgae in response to various environmental stressors. Knowledge on thermoprotective effects of this amino acid on freshwater invertebrates is very scarce. Therefore the aim of our study was to determine the effect of L-PROL at concentrations: 10 mg/L, 20 mg/L and 50 mg/L on swimming behavior (immobilization, swimming track density, swimming speed, turning ability) of Daphnia magna subjected to temperatures: 22 °C, 35 °C and 38 °C. We found that L-PROL elevated all the measured swimming parameters at 22 °C when compared to the untreated crustaceans. Furthermore, L-PROL alleviated heat-induced inhibition of these parameters in the experimental animals subjected to 35 °C. The results suggest that L-PROL stimulates swimming performance and alleviates alterations of swimming parameters induced by heat stress in D. magna. Moreover, these findings may support the hypothesis that in natural conditions, L-PROL may protect crustaceans against thermal stress.