There is substantial evidence supporting a hypertrophic action of serotonin (5-HT) in cardiomyocytes. However, little is known about the mechanisms involved. We previously demonstrated that 5-HT-induced hypertrophy depends, in part, on the generation of reactive oxygen species (ROS) by monoamine oxidase-A (MAO-A). Cardiomyocytes express 5-HT₂ receptors which may also participate in hypertrophy. Here, we analyzed the respective contribution of 5-HT₂ receptors and MAO-A in H9C2 cardiomyoblast hypertrophy. 5-HT induced a dose-dependent increase in ³H-leucine incorporation and stimulation of two markers of cardiac hypertrophy, ANF-luc and SK-actin-luc reporter genes. Experiments using 1 µM 5-HT showed that hypertrophic response occurred independently from MAO-A. Using pharmacological inhibitors (M100907 and ketanserin), we identified a novel mechanism of action involving 5-HT_2A receptors and requiring Ca²⁺/calcineurin/NFAT activation. The activation of this hypertrophic pathway was fully prevented by 5HT_2A inhibitors and was unaffected by MAO inhibition. When 10 µM 5-HT was used, an additional hypertrophic response, prevented by the MAO inhibitors pargyline and RO 41-1049 was observed. Unlike the 5HT_2A-receptor mediated H9C2 cell hypertrophy, MAO-A-dependent hypertrophic response required activation of extracellular-regulated kinases (ERK). In conclusion, our results show the existence of a dose-dependent shift of activation of distinct intracellular pathways involved in 5-HT-mediated hypertrophy of cardiac cells.