Nitric oxide (NO) is a very important molecule taking part in various signaling pathways in plants including plant responses to different stress conditions. In this work we have investigated the effect of NO on the cytosolic free calcium concentration ([Ca²⁺]_cyt) under oxidative stress induced by hydrogen peroxide. Using seedlings of transformed Arabidopsis thaliana expressing Ca²⁺-reporter apoaequorin in cytosol, we have shown that H₂O₂ influences [Ca²⁺]_cyt in a dose-depended manner and induces two peaks of calcium-dependent chemiluminescence measured in plant seedlings. The NO donors sodium nitroprusside (SNP) and (±)-E-2-[(E)-Hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR-1) alone led to a transient increase in [Ca²⁺]_cyt, but simultaneously reduced the amplitude of the first peak of H₂O₂–induced [Ca²⁺]_cyt increase by 42%. The nitric oxide synthase (NOS) and nitrate reductase (NR) inhibitor analysis showed that the production of NO, participating in H₂O₂-induced Ca²⁺-response, occurred due to NOS-like plant enzyme to be sensitive to oxidative stress. In summary, our data indicate that NO is likely to have a protective effect under oxidative stress and reduced a calcium increase induced by reactive oxygen species like hydrogen peroxide.