While the benefit and risk of estrogen replacement therapy for cardiovascular disease remains controversial, women frequently choose alternatives to estrogen such as phytoestrogen for treatment of menopause even though medical indications for estrogens may exist. Phytoestrogens also possess distinct advantages over mammalian estrogens because their usage in men without feminizing side effects. Nevertheless, the cardiac contractile function of estrogen or phytoestrogen has not been clearly elucidated. The aim of the present study was to compare the effect of 17β estradiol (E2) and phytoestrogen α-zearalanol (ZAL) on cardiac mechanical function and intracellular Ca2+ transients at cellular levels. Isolated ventricular myocytes from adult female rats were stimulated to contract at 0.5 Hz. Contractile properties were evaluated using an IonOptix MyoCam® system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (± dL/dt). Intracellular Ca2+ properties were evaluated as fura-2 fluorescent intensity change (ΔFFI) and intracellular Ca2+ decay rate. Acute administration of E2 (10−9–10−5 M) elicited a concentration-dependent increase in PS and ΔFFI, with maximal augmentation of approx 35% and 25%, respectively. TPS, TR90, ± dL/dt, resting intracellular Ca2+ level, and intracellular Ca2+ decay were unaffected by E2. None of the mechanical or intracellular Ca2+ indices tested was affected by phytoestrogen ZAL (10−9–10−5 M). Our results revealed a direct cardiac stimulatory action from E2 but not from phytoestrogen ZAL on ventricular contraction, likely mediated through enhanced intracellular Ca2+ release.