Phytoestrogens exert protective effects on the cardiovascular system through mechanisms that have yet to be clearly demonstrated. The aim of this study was to evaluate the protective effects exerted by genistein on cardiomyoblasts (H9C2) against oxidative stress, nitric oxide (NO) release, viability, proliferation/migration and mitochondrial function. H9C2 cultured in physiological or peroxidative conditions, were treated with genistein in the absence or presence of estrogen receptors (ERs), G protein‑coupled‑estrogenic‑receptors (GPER), Akt, extracellular‑signal‑regulated kinases 1/2 (ERK1/2) and p38 mitogen activated protein kinase (p38MAPK) blockers. Cell viability, proliferation, migration, mitochondrial membrane potential, mitochondrial oxygen consumption and oxidant/antioxidant system, were measured by specific assays. Western blot assay was used for the analysis of NO synthase (NOS) subtypes' and expression and activation of various kinases. In all experiments 17β‑estradiol was used for comparison. The results showed that phytoestrogens and estrogens can increase cell viability, proliferation/migration and improve mitochondrial membrane potential and oxygen consumption of H9C2. Furthermore, NO release was modulated by genistein and 17β‑estradiol. These effects were reduced or abolished by the pre‑treatment with ERs, GPER, Akt, ERK1/2 and p38MAPK blockers. Finally, a reduction of reactive oxygen species production and an increase of glutathione content was found in response to the two agents. In H9C2 cultured in physiological conditions, genistein induced endothelial NOS‑dependent NO production through the involvement of estrogenic receptors and by the modulation of intracellular signalling related to Akt, ERK1/2, and p38MAPK. Moreover, estrogens and phytoestrogens protected H9C2 against oxidative stress by reducing inducible NOS expression and through the modulation of the antioxidant system and mitochondrial functioning.