Sir John Spencer (c. 1549 – 9 January 1600) was an English nobleman, politician, landowner, sheriff, knight, and MP from the Spencer family.
He was born the son of Sir John Spencer (died 1586) of Althorp, Northamptonshire, and his wife Katherine Kitson, daughter of Sir Thomas Kitson of Hengrave, Suffolk. He was educated at Trinity College, Cambridge and trained in the law at the Middle Temple. He succeeded his father in 1586, inheriting estates at Wormleighton, Warwickshire and Althorp, Northamptonshire, and was knighted in 1588.
He was appointed a JP for Bedfordshire in 1577, for Northamptonshire in 1584 and High Sheriff of Northamptonshire for 1578–79 and 1590–91.
He was elected MP for Northampton in 1572.
Spencer died 9 January 1600 and was buried in St Mary the Virgin Church, Great Brington, the parish church for Althorp.
Spencer married Mary, the only daughter and heiress of Sir Robert Catlin, who brought him estates in both Leicestershire and Northamptonshire. They had one son, Robert, who became the first Baron Spencer.
Spencer was a direct ancestor of Lady Diana Spencer.