His father John Bartholomew Senior started a cartographical establishment in Edinburgh,Scotland and he was educated in the work. He was subsequently assistant to the German geographer August Petermann, until in 1856 he took up the management of his father's firm. For this establishment, now known as the Edinburgh Geographical Institute, Bartholomew built up a reputation unsurpassed in Great Britain for the production of the finest cartographical work.

Bartholomew is best known for the development of colour contouring (or hypsometric tints), the system of representing altitudes on a graduated colour scale, with areas of high altitude in shades of brown and areas of low altitude in shades of green. He first showcased his colour contouring system at the Paris Exhibition of 1878; although it initially met with skepticism, it went on to become standard cartographical practice.

Among his numerous publications, particularly worthy of note is the series of maps of Great Britain reduced from the Ordnance Survey to scales of ½ inch and ¾ inch to 1 mile, with relief shown by contour lines and hypsometric tints. The ½ inch series is among the finest of its kind ever produced.

Upon his retirement in 1888, John Bartholomew was succeeded in the firm by his son John George, who extended the ½ inch series, and applied its principles to many other works. Bartholomew died in London on 29 March 1893.