(−)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined its effects on the acidosis-induced change in the Ca2+ sensitivity of myofilaments in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. EGCg (0.1 mM) significantly inhibited the decrease caused by lowering the pH from 7.0 to 6.0 in the Ca2+ sensitivity of myofibrillar ATPase activity in cardiac muscle, but not in skeletal muscle. Studies on recombinant mouse cardiac troponin C (cTnC) and chicken fast skeletal troponin C (sTnC) using circular dichroism and intrinsic and extrinsic fluorescence spectroscopy showed that EGCg bound to cTnC with a dissociation constant of ∼3–4 μM, but did not bind to sTnC. By presumably binding to the cTnC C-lobe, EGCg decreased Ca2+ binding to cTnC and overcame the depressant effect of protons on the Ca2+ sensitivity of the cardiac contractile response. To demonstrate isoform-specific effects of the action of EGCg, the pH sensitivity of the Ca2+ response was examined in cardiac myofibrils in which endogenous cTnC was replaced with exogenous sTnC or cTnC and in skeletal myofibrils in which the endogenous sTn complex was replaced with whole cardiac Tn complex (cTn). The results suggest that the binding of EGCg to the cardiac isoform-specific TnC or Tn complex alters the effect of pH on myofilament Ca2+ sensitivity in striated muscle.