In ocean thermal energy conversion (OTEC) systems, the initial working fluid charging amount is crucial as it relates to the system’s performance and economic cost. But currently, there is no clear standard for it, and as the working fluid circulating pump is the most direct and only means to regulate the working fluid flow, it is necessary to investigate the coupled effects of it and the working fluid charging amount on the system performance. To this issue, a corresponding experimental facility was established. The coupled effects were investigated by energy and exergy analysis under varying temperature difference. The results showed that varying working fluid charging amount changed the physical state of the working fluid, and the effect on the system varied under dissimilar temperature difference conditions. Compared to the standard charge (8 kg), 75 % of the standard charging amount diminished the system thermal efficiency by 17 % and exergy efficiency by 16 % at small temperature difference (20 °C), whereas a charging amount of more than 100 % resulted in improved thermal efficiency by 14 % and exergy efficiency by 13 %. At large temperature difference (28 °C), 75 %, 125 % and 150 % of the standard working fluid charging amount all improved system performance (thermal efficiency by maximum 16 %, exergy efficiency by maximum 21 %). Besides, an increase in circulating pump frequency improved system performance regardless of the temperature difference and working fluid charging amount. This research innovatively investigates the above problems in the application scenario of OTEC, and provides theoretical and data support for the development of OTEC technology.