MacGregor was born in Gravesend to Major Duncan MacGregor, a Scottish soldier, and Elizabeth, the daughter of a Baronet. When he was two weeks old, he was amongst the survivors of the sinking of Kent. His schooling followed his father's postings, and he attended The King's School in Canterbury and Trinity College in Dublin. He graduated from Trinity College, Cambridge, having shown a talent for mathematics. He was known by his peers for his strong Christian faith and although he had aspired to be a missionary and also considered civil engineering, his scientific interests led him to London to study for the Bar, specialising in patent law. During his time in the capital, he became heavily involved with philanthropy and was instrumental in launching the Ragged School movement's Shoeblack brigade. He also became involved with open air missions and was also able to follow his passion for travel. In 1849 he spent nine months exploring southern Europe, Palestine and Egypt. He was introduced to canoeing during a trip to the United States and Canada in 1858.
Boat designer
MacGregor was a champion marksman but turned to boating when a railway accident left him unable to hold a rifle steady. The boat he designed was a 'double-ended' kind of canoe inspired by the Northern Americankayaks, but built in Lambeth of lapstrake oak planking, decked in cedar covered with rubberised canvas with an open cockpit in the center. It measured 15 feet long, 28 inches wide, nine inches deep and weighed 80 pounds and was designed to be used with a double-bladed paddle. He named the boat Rob Roy after the celebrated Scottish outlaw of the same name, to whom he was related. During the 1860s, he had at least seven similar boats built and he sailed and paddled them in Europe, the Baltic and the Middle East. One of those canoes is now based at the National Maritime Museum Cornwall. The version he used for his expedition to Egypt, Syria and Palestine in 1868/69 was slightly smaller but was designed so that he could sleep in it. He was accompanied by a dragoman, Hany, and two retainers who maintained the various base camps on the journey. He visited the Nile Delta, the rivers of Damascus, as well as parts of the River Jordan and Kishon River. Whilst exploring the water courses above Lake Hula he was taken prisoner by villagers from Al-Salihiyya who lifted both him and the Rob Roy canoe out of the river whilst he kept them at bay with his paddle. He was responsible for the first scientific survey of the area.
Writings
In 1866, he published A Thousand Miles in the Rob Roy Canoe, which popularised the design and, more importantly, the concept: "in walking you are bounded by every sea and river, and in a common sailing-boat you are bounded by every shallow and shore; whereas,...a canoe be paddled or sailed, or hauled, or carried over land or water". The book was internationally successful; with subsequent books and public appearances, it earned MacGregor more than ten thousand pounds. Scottish author Robert Louis Stevenson's 1876 voyage by canoe through the canals and rivers of France and Belgium, published in 1878 as An Inland Voyage, used "Rob Roy" canoes. made by John MacGregor from data gathered during his visit in January 1869.