The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1–10 μM for 6–24 h.