The aim of the present study was to evaluate the in vitro cytotoxicity of self-adhesive dual-cured resin cement (SADRC) polymerized beneath three different cusp inclinations of zirconia with different light curing time. A commercial SADRC (Multilink Speed) was polymerized beneath zirconia (ZrO2) with three different cusp inclinations (0°, 20°, and 30°) for 20 s or 40 s. After being stored in light-proof box for 24 h, the ZrO2-SADRC specimens were immersed in DMEM for 72 h and then we got the extract solution, cultured the human gingival fibroblasts (HGF, 8 × 103 per well) with 100% or 50% concentrations of the extract solution for 24 h, 72 h, and 120 h, respectively, and evaluated cytotoxicity of the polymerized SADRC with CCK-8 assay in optical density (OD) values, relative growth rates (RGR), and cytotoxicity grades. Statistical analysis was conducted using a two-way ANOVA followed by post hoc Student-Newman-Keuls test. The OD values varied from 0.8930 to 3.2920, the RGR varied from 33.93% to 98.68%, and the cytotoxicity grades varied from 0 to 2. There was significant difference in the OD values among the different cusp inclinations of zirconia (P < 0.001), and there was significant difference in the OD values between the different light curing times in some situations (P < 0.05). The cusp inclination of zirconia affects the in vitro cytotoxicity of SADRC. Prolonging the light curing time from 20 s to 40 s can reduce the in vitro cytotoxicity of SADRC when the cusp inclination of zirconia is smaller than 20°.