Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10−12, 10−9, 10−4 m; Experiment 4: 0, 10−3 m), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat-stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10−4 m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non-HSO without melatonin. Melatonin did not have any effect in embryos from non-HSO groups compared with the control. In Experiment 4, 10−3 m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non-HSO when compared to melatonin-untreated oocytes/embryos. In conclusion, 10−4 m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.