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Napier MacNab was born in Newark (Niagara-on-the-Lake), Ontario,to Allan MacNab, lieutenant in the 71st Regiment and the Queen's Rangers under Lt-Col. John Graves Simcoe, and Anne, daughter of Capt. Peter William Napier, R.N., the commissioner of the port and harbour of Quebec. After the Queen’s Rangers were disbanded the family moved around the country in search of work and eventually settled in York (Toronto) where MacNab was educated at the Home District Grammar School.
As a fourteen year old boy he fought in the War of 1812. 
In 1826 he moved from York (Toronto) to Hamilton, Ontario where he established a successful law office, though it was chiefly through land speculation that he made his fortune. In 1830 he was elected to represent the city in the Legislative Assembly, a position he held for some 27 years.
As a member of the legislature he opposed the reform movement in Upper Canada led by William Lyon Mackenzie. When Mackenzie led the Upper Canada Rebellion in 1837, MacNab was part of the British militia that moved against Mackenzie at Montgomery's Tavern in Toronto on December 7, dispersing Mackenzie's rebels in less than an hour.
MacNab then led a militia of his own against the rebels marching towards Toronto from London, led by Charles Duncombe. Duncombe's men also dispersed when they learned MacNab was waiting for them. In 1838 he was knighted for his zeal in suppressing the rebellion. He served in the Legislative Assembly of the Province of Canada, leading the province from 1854 to 1856. He was elected to the Legislative Council in 1860 representing Western division and served until his death.
A successful entrepreneur as well as politician, MacNab, with Glasgow merchant Peter Buchanan, was responsible for the construction of the Great Western Railway.
MacNab was married twice, first to Victoria Brooke, who died 5 November 1826, possibly of complications following childbirth. Together they had two children. His second marriage to Mary Stewart also ended in tragedy when she died 8 May 1846; they also had two daughters.
His stately 72 room home in Hamilton, Dundurn Castle, is now open to the public. Hamilton's MacNab Street is named after him.