Chesterfield Borough Council says 'essential' work being carried out on the Donut car park today relates to the Northern Gateway masterplan.
A business enterprise centre will eventually be built on part of the car park for start-up and small and medium sized companies.
The centre, which is expected to open in 2019, is a key part of the revised Northern Gateway project.
A Chesterfield Borough Council spokesman said: "This essential work is to do with the business enterprise centre which will be built on part of the island. Site investigations will be taking place.
"The car park will be closed for 24 hours until 7am tomorrow.
"Work will also be carried out at Saltergate multi-storey car park, although it will remain open to users."
Councillor Steve Brunt, cabinet member for town centres and visitor economy, added: "I'm sorry that we will inconvenience some people who regularly use the Donut car park while this work is being carried out.
"We have worked to minimise the disruption as much as possible by planning the work over one 24 hour period and there will be plenty of spaces available in other car parks in the town centre."
The £19.9million Northern Gateway development - which promises to improve a main entrance to Chesterfield and create nearly 500 new jobs - also includes a revamp of the former Co-op building, a full refurbishment of the Saltergate multi-storey car park and environmental and street improvements to Elder Way and part of Knifesmithgate, including wider pavements to create a terrace area.
The Northern Gateway scheme will be funded by a Sheffield City Region Infrastructure Fund grant of £5.83m and £3.5m from the borough council, with the £10.5m Co-op redevelopment being paid for by the private sector.
A rethink of the original plans for the Northern Gateway began in 2015 after it became clear that no large food retailer could be attracted to the site, which was necessary to make the rest of the scheme viable.
The original plan also included proposals for town centre housing. While this is not in the revised plan, it remains an option for the future.