Temporary sequential biventricular pacing (BiVP) is a promising treatment for postoperative cardiac dysfunction, but the mechanism for improvement in RV dysfunction is not understood. In the present study, cardiac output (CO) was optimized with sequential BiVP in six anesthetized, open-chest pigs during control and acute right ventricular pressure overload (RVPO). Ventricular contractility was assessed by the maximum rate of increase of ventricular pressure (dP/dt_max). Mechanical interventricular synchrony was measured by the area of the normalized right-left ventricular (RV-LV) pressure diagram (A_PP). Positive A_PP indicates RV pressure preceding LV pressure, while zero indicates complete synchrony. In the control state, CO was maximized with nearly simultaneous stimulation of the RV and LV, which increased RV (p=0.006) and LV dP/dt_max (p=0.002). During RVPO, CO was maximized with RV-first pacing, which increased RV dP/dt_max (p=0.007), but did not affect LV dP/dt_max , while also decreasing the left-to-right end-diastolic pressure gradient (p=0.023). Percentage increase of RV dP/dt_max was greater than LV dP/dt_max (p=0.014). There were no increases in EDP to account for increases in dP/dt_max . In both control and RVPO, RV dP/dt_max was linearly related to A_PP (r=0.779, p<0.001). The relation of CO to A_PP was curvilinear, with a peak in CO. This peak occurred with positive A_PP in the control state (p=0.004) and with A_PP approaching zero during RVPO (p=0.001). These observations imply that in our model, BiVP optimization improves CO by augmenting RV contractility. This is mediated by changes in mechanical interventricular synchrony. Afterload increases during RVPO exaggerate this effect, making CO critically dependent on simultaneous pressure generation in the RV and LV, with support of RV contractility by transmission of LV pressure across the interventricular septum.