In 1825 John Stevens Henslow, Charles Darwin's teacher at Cambridge, succeeded Martyn as professor of botany and soon realized that a larger site, farther from the centre of Cambridge, was desirable for the Botanic Garden. In 1831 the University purchased the present site of about 40 acres to the south of the town on the Trumpington Road, and in 1846 the first tree was planted. It had been the intention to lay out the whole 40 acres as a Botanic Garden, but presumably funds were lacking, and in fact only 20 acres were planted, the remainder being let out as allotments.
The planning of the new Garden was carried out by Professor Henslow, assisted by young Cardale Babington. The land was flat and unpromising as a garden site, but the layout was planned with great skill, utilizing an old gravel pit to construct a lake with a high mound running into it. Trees and shrubs were planted according to their botanical sequence, a range of glasshouses was built in the 1860s, and a rock garden, one of the earliest of its kind in the country, was constructed about the same time. The Garden has also long been known for its many fine specimens of rare trees. By the 1870s the main features of the Garden had been developed and, it was ready to play its part in the great expansion of botanical teaching and research that was about to take place at Cambridge. During the early years of the 20th century much of the pioneer work of William Bateson, Charles Chamberlain Hurst, and Edith Rebecca Saunders on plant genetics was carried out at the Garden, and it was later used for researches on plant physiology by Frederick Blackman and George Edward Briggs, and on plant pathology by Frederick Tom Brooks and others. In 1921 the University appointed H. Gilbert-Carter as the first scientific Director of the Garden.