Pumping ventilation (PV), a special single-sided ventilation (SSV), has been certified as an effective strategy to improve the air exchange rate of SSV. However, most studies targeted on the single space, and few studies have been focused on the effect of internal partitioning on PV. This paper aims to evaluate the ventilation performance of PV influenced by different configurations of internal partitioning. Computational fluid dynamics (CFD) simulation was used to predict the flow fields and ventilation rates. The width (w/H), height (h/H) and location (d/H) are the three main internal partition parameters considered in this study. The simulation results showed that the total, mean and fluctuating ventilation rates all decrease with wider internal partitions. The normalized total ventilation rate decreases by 7.6% when w/H is increased from 50% to 75%. However, the reduction rate is only 0.23% between w/H = 0 and 25%, and only 0.61% between w/H = 25% and 50%. The ventilation rate is hardly reduced by increasing the partition width when w/H < 50%, whereas greatly reduced by wider partition for w/H > 50%. Increasing the partition height will reduce the mean ventilation rate but promote the fluctuating and total ventilation rate in some cases. An increase of total ventilation rate by 1.4% is observed from h/H = 50% to 75%. The ventilation rate is larger when the internal partition is attached to the leeward or windward wall. The total, mean and fluctuating ventilation rates for d/H = 50% are relatively higher than d/H = 0 by 1.5%, 3.1% and 0.8%, respectively. Hence the internal partition should be mounted attached to the windward wall so as to obtain the greatest pumping ventilation rate. The periodicity of pumping flow oscillation and pumping frequency are independent of the partition configurations. The peak power of pumping flow is the lowest for the widest internal partition and is negatively affected by the partition height, but it generally has a positive correlation with the distance between the partition and leeward wall. Present research will help to understand pumping ventilation mechanism in real buildings with internal partitioning and provide theoretical basis for developing unsteady natural ventilation technology in low-carbon buildings.