Over 150,000 people joined the chain which extended from the historic city of Durango to Pamplona, the capital of the neighbouring Navarra region which has a significant Basque-speaking population, according to the event organiser called "It's In Our Hands".
Demonstrators, many draped in red, white and green Basque flags, raised their linked hands in the air along country roads and through towns in the Basque region as a helicopter flew over.
At one point along the route participants snaked along a large white canvas with the message "Basques Decide" written in large yellow letters in English that was set on the pavement of a parking lot.
Demonstrators also wrote "Gure Erabaki", which means "Our Decision" in the Basque language, and "Gure Eskubidea", which means "Our Right", in large white letters on green hills along the route.
The protest was backed by tiny Basque separatist parties and it was inspired by a similar event held in September in Catalonia when hundreds of thousands of people showed their support for independence by joining hands along a 400-kilometre stretch of the Mediterranean coast.
The Basque Country, which has its own distinct language and culture, has long sought – and won – greater powers for itself, especially in areas such as taxing, education and policing.
A peaceful Basque independence movement has gathered pace in recent months, partly inspired by large pro-independence demonstration in Catalonia.
The regional government of Catalonia is pushing to hold a referendum on independence from Spain on November 9, flying in the face of fierce opposition from the central government in Madrid.
The armed Basque separatist group ETA, which has been weakened by a string of arrests and dwindling popular support for its violent tactics, announced an end to violence.
Earlier this year the group, which is blamed for the deaths of 829 people in a four-decade campaign for an independent Basque homeland in northern Spain and southwestern France, said it would put its arsenal of weapons "out of operational use".
But it has so far refused to disband or disarm as demanded by the Spanish and French governments.