Oxidative stress during the in vitro production of embryos culminates with a rise in reactive oxygen species (ROS) levels and apoptosis. Menadione (MD) induces ROS generation and can be used as a tool to understand the actions of oxidative stress on embryo development. This study evaluated the effects of different MD concentrations during in vitro culture on bovine embryo development, intracellular levels of ROS and apoptosis rate. Cumulus-oocyte complexes were matured in vitro in TCM-199 with bicarbonate, hormones and 10% FCS during 22 h at 38.5°C in 5% CO₂ in air. After fertilization, zygotes were cultured in SOF medium at 38.5°C in 5% CO₂ in air, for 7 d (IVF = Day 0). On Day 6, SOF was supplemented with 0 (Control group), 2.5 μM MD (MD 2.5) or 5.0 μM MD (MD 5.0), The cleavage and blastocyst rates were evaluated at Days 3 and 7, respectively. Day-7 blastocysts were stained with H₂DCFDA (Molecular Probes, Invitrogen) or with TUNEL (in situ cell death detection kit, Roche Life Science) to evaluate the ROS levels and the apoptotic rates, respectively. Stained embryos were evaluated under epifluorescence (excitation 495/510–550 nm and emission 520/590 nm, respectively for H₂DCFDA and TUNEL) and the images were analyzed by Q-Capture Pro Image Software to determine the fluorescence intensity. Data were analyzed by ANOVA followed by Tukey’s test (P < 0.05) and are presented as Mean ± SEM. The cleavage rates were similar (P > 0.05) among groups (78.2% ± 1.14 to 80.5% ± 0.88). The blastocyst rates were lower (P < 0.05) in MD 5.0 (25.9% ± 2.06^b) compared with Control (36.4% ± 1.51^a), and both were similar (P > 0.05) to MD 2.5 (32.5% ± 1.94^ab). The ROS levels were higher in MD 2.5 (0.75 ± 0.05^b) and MD 5.0 (1.11 ± 0.07^b) compared with Control (0.72 ± 0.04^a). The rates of apoptosis were higher (P < 0.05) in MD 5.0 (19.24% ± 0.69^c) compared with MD 2.5 (13.28% ± 0.71^b) and Control (10.0% ± 0.45^a). In conclusion, MD concentrations of 2.5 and 5.0 μM were effective in inducing oxidative stress in bovine embryos produced in vitro and the detrimental effects were dose-dependent. The higher the oxidative stress, the more detrimental were the effects, causing reduction in the embryonic development, increasing on the intracellular ROS levels and apoptosis.