Lightfoot was born in Liverpool, where his father John Jackson Lightfoot was an accountant. His mother, Ann Matilda Barber, was from a family of Birmingham artists. He was educated at King Edward's School, Birmingham, under James Prince Lee. His contemporaries included Brooke Foss Westcott and Edward White Benson. In 1847, Lightfoot went to Trinity College, Cambridge, and read for his degree along with Westcott. He graduated senior classic and 30th wrangler, and was elected a fellow of his college. From 1854 to 1859 he edited the "Journal of Classical and Sacred Philology". In 1857, he became tutor and his fame as a scholar grew. He was made Hulsean professor in 1861, and shortly afterwards chaplain to the Prince Consort and honorary chaplain in ordinary to Queen Victoria.