Channelrhodopsin-2 (ChR2)-based optogenetic technique has been increasingly applied to cardiovascular research. However, the potential effects of ChR2 protein overexpression on cardiomyocytes are not completely understood. The present work aimed to examine how the doxycycline-inducible lentiviral-mediated ChR2 expression may affect cell viability and electrophysiological property of neonatal rat ventricular myocyte (NRVM) cultures. Primary NVRMs were infected with lentivirus containing ChR2 or YFP gene and subjected to cytotoxicity analysis. ChR2-expressing cultures were then paced electrically or optically with a blue light-emitting diode, with activation spread recorded simultaneously using optical mapping. Results showed that ChR2 could be readily transduced to NRVMs by the doxycycline-inducible lentiviral system; however, high-level ChR2 (but not YFP) expression was associated with substantial cytotoxicity, which hindered optical pacing. Application of bromodeoxyuridine significantly reduced cell damage, allowing stimulation with light. Simultaneous optical Vm mapping showed that conduction velocity, action potential duration, and dVm/dtmax were similar in ChR2-expressing and control cultures. Finally, the ChR2-expressing cultures could be optically paced at multiple sites, with significantly reduced overall activation time. In summary, we demonstrated that inducible lentiviral-mediated ChR2 overexpression might cause cytotoxicity in NRVM cultures, which could be alleviated without impairing electrophysiological function, allowing simultaneous optical pacing and Vm mapping.