The aim of this study was to investigate the effects of nitric oxide (NO) and reactive oxygen species (ROS) on L-type calcium channel (LTCC) gating properties of cardiomyocytes during long-term isoproterenol (ISO) stimulation. Expression and activity of nNOS as well as S-nitrosylation of LTCC α1C subunit significantly decreased in the myocardium of SUS rats. Long-term ISO stimulation increased ROS in cardiomyocytes of SUS rats. ISO-enhanced calcium current (ICa,L) in the SUS group was less than that in the CON group. The maximal ICa,L decreased to about 80% or 60% of initial value at the 50th minute of ISO treatment in CON or SUS group, respectively. Specific inhibitor NAAN of nNOS reduced maximal ICa,L to 50% of initial value in the CON group; in contrast, NO donor SNAP maintained maximal ICa,L in SUS group to similar extent of CON group after 50 min of ISO treatment. Long-term ISO stimulation also changed steady-state activation (P<0.01), inactivation (P<0.01), and recovery (P<0.05) characteristics of LTCC in SUS group. In conclusion, NO-induced S-nitrosylation of LTCC α1C subunit may competitively prevent oxidation from ROS at the same sites. Furthermore, LTCC can be protected by NO during long-term ISO stimulation.