There is limited research into horse slaughter, particularly ante mortem welfare, and the effectiveness of captive-bolt gun (CBG) stunning, despite this being a widely used method worldwide. To address this evidence gap and explore associations between ante and post mortem factors, the welfare of 62 horses was assessed at a commercial Italian abattoir. Animal-based measures were used to identify stress-related behaviours and stunning effectiveness. A sub-sample (44%; 27/62) of heads were assessed for gross brain pathology. All animals in the study showed stress-related behaviours at all stages of the slaughter process. Additionally, 53% (33/62) of horses slipped in the stunning box, with poor floor surface condition and use of force associated with this. At least one sign of an ineffective stun was observed in 22% (14/62) of animals. Six animals were shot twice, with the application of a second shot significantly associated with a higher stress behaviour index score in the stunning box. Damage to critical brainstem structures was found in 85% (23/27) of heads that were assessed with gross pathology. An absence of damage to critical brainstem structures meant that animals were ten times more likely to show signs of ineffective stunning. These results highlight the risks to equine welfare throughout the slaughter process and suggest that mitigating ante mortem stress could improve stunning effectiveness, whilst CBG usage should be refined to ensure that critical brainstem structures are targeted.