Serotonin, 5-hydroxytryptamine (5-HT), plays various roles in the fruit fly, Drosophila melanogaster. Previous studies have shown that 5-HT modulates the heart rate in third instar larvae. However, the receptor subtypes that mediate 5-HT action in larval cardiac tissue had yet to be determined. In this study, various 5-HT agonists and antagonists were employed to determine which 5-HT receptor subtypes are responsible for the positive chronotropic effect by 5-HT. The pharmacological results demonstrate that a 5-HT2B agonist significantly increases the heart rate; however, 5-HT1A, 5-HT1B, and 5-HT7 agonists do not have a significant effect on the heart rate. Furthermore, 5-HT2 antagonist, ketanserin, markedly reduces the positive chronotropic effect of 5-HT in a dose–response manner. Furthermore, we employed genetic approaches to confirm the pharmacological results. For this purpose, we used RNA interference line to knock down 5-HT2ADro and also used 5-HT2ADro and 5-HT2BDro insertional mutation lines. The results show that 5-HT2ADro or 5-HT2BDro receptor mutations reduce the response of the heart to 5-HT. Given these results, we conclude that these 5-HT2 receptor subtypes are involved in the action of 5-HT on the heart rate in the larval stage.