The Headstart Pre School Centre in Preston beat around 300 other entrants to win the accolade, voted for nationally by parents.
The nursery, set up in 1989 when the colleagues borrowed a building from their local council, established its own centre for staff training in 1991.
Headstart chief executive Anne Wynne-Jones said she was "gobsmacked" by the award. The nursery will spend the 2,000 cash prize on establishing a soft play area for babies.
Prizes also went to Oakfield Nursery School in Altrincham, and Granby House Nursery in Parkgate, Rotherham.
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