One of the major restrictions on the production of adenoviral vectors (AdV) is due to the decrease in virus concentration concomitant with an increase in cell concentration at infection (CCI) which is known as "cell density effect", this could be because of the limited access to the nutrients or significant accumulation of toxic by-products. However, current strategies, such as developing robust serum-free medium and performing nutrient feeding, will partially address this issue. Therefore, the objective of this study was to further optimize serum-free culture medium by exploring the influence of calcium ion on virus production. Our studies showed that an optimal concentration of calcium ion significantly enhances AdV production, especially at a high CCI. During the virus infection process, a high concentration of calcium ion (${geq}$ 1 mM) caused a reduction in virus infection efficiency, possibly as a result of cell aggregation. However, by optimizing the timing of the addition of calcium ion (i.e., 12 hours post-infection [hpi]), high virus infection efficiency could be maintained. The addition of 0.1 to 2 mM calcium ion at 12 hpi increased virus proliferation dose-dependently. Therefore, the optimal selection of both the concentration and the supplementation time of calcium ion during the process of AdV production could minimize the "cell density effect," and a 2.6- and 3.2-fold increase in virus concentration could be achieved at CCI3 and CCI4, respectively.