5-HT has been reported to possess significant effects on cardiac activities, but activation of 5-HTR on the cell membrane failed to illustrate the controversial cardiac reaction. Because 5-HT constantly comes across the cell membrane via 5-HT transporter (5-HTT) into the cytoplasm, whether 5-HTR is functional present on the cellular organelles is unknown. Here we show 5-HTR3 and 5-HTR4 were located in cardiac mitochondria, and regulated mitochondrial activities and cellular functions. Knock down 5-HTR3 and 5-HTR4 in neonatal cardiomyocytes resulted in significant increase of cell damage in response to hypoxia, and also led to alternation in heart beating. Activation of 5-HTR4 attenuated mitochondrial Ca2+ uptake under the both normoxic and hypoxic conditions, whereas 5-HTR3 augmented Ca2+ uptake only under hypoxia. 5-HTR3 and 5-HTR4 exerted the opposite effects on the mitochondrial respiration: 5-HTR3 increased RCR (respiration control ratio), but 5-HTR4 reduced RCR. Moreover, activation of 5-HTR3 and 5-HTR4 both significantly inhibited the opening of mPTP. Our results provided the first evidence that 5-HTR as a GPCR and an ion channel, functionally expressed in mitochondria and participated in the mitochondria function and regulation to maintain homeostasis of mitochondrial [Ca2+], ROS, and ATP generation efficiency in cardiomyocytes in response to stress and O2 tension.