The results obtained from this study suggest that a period of separation and car travel are stressful events for dogs, with travel in a vehicle eliciting a more pronounced stress response. Further, a single dose of 4 mg/kg CBD 2 h prior to exposure to these events attenuates some indicators of acute canine stress, which is likely to improve canine emotional wellbeing. Additional research is required to better understand the effect of CBD at other dosages, formulations, and whether cumulative administration improves efficacy.