Gut-derived factors in intestinal lymph have been shown to trigger myocardial contractile dysfunction. However, the underlying cellular mechanisms remain unclear. We examined the effects of physiologically relevant concentrations of mesenteric lymph collected from rats with 40% burn injury (burn lymph) on excitation-contraction coupling in rat ventricular myocytes. Burn lymph (0.1-5%), but not control mesenteric lymph from sham-burn animals, induced dual positive and negative inotropic effects, depending upon the concentrations used. At lower concentrations (<0.5%), burn lymph increased the amplitude of myocyte contraction (1.6 ± 0.3-fold, n=12). At higher concentrations, (>0.5%), burn lymph initially enhanced myocyte contraction, which was followed by a block of contraction. These effects were partially reversible upon washout. The initial positive inotropic effect was associated with a prolongation of action potential duration (APD₉₀, 2.5 ± 0.6-fold, n=10), leading to significant increases in the net Ca²⁺ influx (1.7 ± 0.1-fold, n=8). There were no significant changes in the resting membrane potential. The negative inotropic effect was accompanied by a decrease in the action potential plateau (overshoot decrease by 69 ± 10%, n=4) and membrane depolarization. Voltage-clamp experiments revealed that positive inotropic effects of burn lymph were due to an inhibition of the transient outward K⁺ currents (I_to) that prolong action potential duration, and the inhibitory effects were due to a concentration-dependent inhibition of Ca²⁺ currents (I_Ca) that lead to a reduction of action potential plateau. These burn lymph-induced changes in cardiac myocyte Ca²⁺ handling can contribute to burn-induced contractile dysfunction and ultimately to heart failure.