One elephant died and four others were injured on Monday when the lorry that was carrying them overturned.
The accident happened on the A-30 motorway towards Murcia in Albacete province. The animals were being transported by a circus troupe after a performance nearby.
Sources said that the lorry appeared to lose control after overtaking and one of the animals was killed on impact while the other wandered the carriageway until rescued.
Animal rights groups protested and called for circuses with live animals to be banned.