Simple SummaryThe mixing of bovine faeces and urine leads to climate-damaging ammonia emissions. If cattle could be taught to use a latrine, this would reduce the area of emissions, the separation of excreta could easily be accomplished by mechanical means, and animal health could be improved. Attempts to train toileting in cattle have shown limited success. In children, toileting is trained mainly by (i) interrupting voiding that starts outside the toilet, then taking the child to the toilet and rewarding the resumption of excretion, or (ii) placing the child on the toilet, waiting for urination/defecation and rewarding appropriate excretory behaviour. The first method is reported to be more successful. Thus, a similar procedure was evaluated for training latrine use for urination in calves. On 95% of occasions, the calves inhibited or stopped urination when receiving a signal to move to the latrine, and on 65% of occasions, they reinitiated urination in the latrine. Furthermore, during 63% of urinations in the latrine, the calves oriented towards the reward location before any food was delivered, providing additional evidence that calves can be successfully toilet trained with food rewards.Untrained cattle do not defecate or urinate in defined locations. The toilet training of cattle would allow urine and faeces to be separated and stored, reducing climate-damaging emissions and improving animal health. In a proof-of-concept study, we evaluated a novel protocol for toilet training in cattle. Five heifer calves (and yoked controls) were trained in the voluntary (operant) behaviours of a toileting chain. Then, reflexive urinating responses were incorporated into the chain, with toileting signalled by a tactile (vibratory) stimulus. On 95% of occasions, the calves inhibited/interrupted urination when receiving the stimulus, and on 65% of these occasions, reinitiated urination in the latrine. Furthermore, during 63% of urinations in the latrine, the calves oriented to the reward location before any food was delivered, providing additional evidence that calves can be successfully toilet trained with food rewards. Yoked controls failed to learn most of the operant elements and all the reflexive responses of toilet training. The results show that reflexive behaviours can be incorporated into voluntary toileting sequences with cattle and extend the range of species that can be toilet trained. Future refinement of the protocol to allow training under practical farm conditions offers the potential to mitigate climate damage and improve animal health.