This study examines the effects of bismuth oxide (Bi2〇3) radiopacifier on the physicochemical properties and in vitro osteogenic activities of sol-gel-derived dicalcium silicate cements. The setting time, diametral tensile strength, radiopacity, solubility, pH value, and phase composition of the cements containing various amounts of Bi2〇3 (5, 10, and 20 wt%) were evaluated and compared to those of ProRoot white-colored mineral trioxide aggregate (WMTA). The osteogenic activities, such as proliferation, differentiation, and mineralization, of MG63 human osteoblast-like cells on cements with and without Bi2〇3 were also examined. The results show that the setting time increased significantly (P < 0.05) with increasing Bi2〇3 content. The pH value and diametral tensile strength of the cements were slightly affected by the introduction of Bi2〇3. After the addition of 5, 10, and 20 wt% Bi2〇3, the radiopacity of the cements became significantly (P < 0.05) higher, with values equivalent to 3.3, 5.8, and 8.4 mm of Al, respectively, which are all greater than the 3 mm of Al recommended by ISO 6876: 2001 standards. The solubility of the three radiopaque cements ranged between 0.8% and 1.1%, which is significantly (P < 0.05) lower than that of WMTA (1.4%). 20 wt% Bi2〇3 led to lower cell proliferation, differentiation, and calcium deposits of MG63 cells on the cement surfaces at all culture times compared those obtained with the other cements. The addition of 10 wt% Bi〗〇3 to dicalcium silicate cement improves the setting time, radiopacity, and osteogenic activity, making the cement a potential alternative to WMTA as a root-end filling material.