One of the elephants died from injuries that it sustained, while two others were hurt, local police say.
The injured animals had to be lifted by a crane from the carriageway.
The incident occurred on Monday afternoon in the town of Pozo Cañada near Albacete. The driver of the truck involved in the crash was not injured.
The vehicle is believed to be a circus lorry.
The director of the government department responsible for Spain's road transport network, Gregorio Serrano, posted a video of the elephant evacuation to social media.
Local police in Albacete released photographs showing three of the five elephants milling around in the lanes of the motorway after the accident.
Mr Serrano said on Twitter that the cause of the crash is still under investigation by the local authorities.
The Pozo Cañada council posted a video of the four surviving elephants in a compound on Sunday evening.
It said (in Spanish) that they are being looked after by the local authority until they can be transferred.
