Description: This study aimed to explore the changes in calcium transient in the development of heart failure and the effects of levosimendan (LeV) on intracellular calcium dynamics. Cultured neonatal rat ventricular myocytes were divided into four groups: normal, norepinephrine (NE) only (10 µmol/L), NE + LeV1 (0.1 µmol/L), and NE + LeV2 (1 µmol/L). The calcium transients of the myocytes loaded with Fluo-3/AM were observed using a laser scanning confocal microscope. Compared with the control group, the calcium wave in the NE group dispersed, propagated slowly, and exhibited dyssynchrony of Ca 2+ release. Norepinephrine accelerated the beating rate of the cultured myocytes, decreased the systolic peak Ca 2+ , and increased the time to peak (Ttp) and decay time (Tau) of calcium transient. Levosimendan increased the synchrony of calcium transient, and reduced Ttp and Tau. In contrast, LeV did not affect the beating rate and systolic peak Ca 2+ . Both NE-only- and LeV-treated groups did not affect resting Ca 2+ and calcium transient amplitude of the myocytes. The currents from L-type calcium channel currents did not differ among the groups. Both NE and LeV shortened the action potential duration, but the effect of the latter was more serious than that of the former. Western blot results showed that the sarco/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2) expression decreased in the NE group but increased in the LeV groups. The sodium–calcium exchanger 1 (NCX1) expression increased in the NE group but decreased in the LeV groups. Long-term exposure to NE decreased myocardial contractility by reducing the peak Ca 2+ of calcium transient and by prolonging and disrupting the conduction of calcium waves. Levosimendan elicits a positive inotropic effect by accelerating the velocity of calcium signal propagation and synchronizing calcium release without increasing calcium influx.