The Glenboro area was visited early in western exploration, possibly as early as the 1730s when the La Verendryes travelled to the Missouri and beyond. History records that the explorer and geographer David Thompson passed through the area in 1798 and made note of the quality of soil. The first permanent European settlers of the area (Jonas Christie and James Duncan) arrived in 1879, and after the federal Homestead Act passed in 1880, many more settlers arrived in the area, including a number of Icelandic settlers between 1889 and 1894.

The arrival of the CPR in 1886 prompted Christie and Duncan to offer land for a town site. The Queens Hotel was the first permanent building of the community, constructed in 1881, and survives into the 21st century. In 1950 the community was formally incorporated as a village.

Steam riverboats were in use on the Assiniboine River between 1875 and 1885. The bow timbers of the last of this "prairie navy" are preserved in a park in Glenboro. The SS Alpha ran aground on April 27, 1885. By this time the advent of the railway had displaced riverboats for transport of passengers, HBC trade goods, agricultural supplies, and grain between Southern Manitoba communities and larger centers such as Winnipeg. A 1904 water tower was installed for watering steam locomotives. The tower was declared both a federal and a provincial heritage site in September 1996. One of the few remaining water towers in Canada, it was destroyed by fire in the early morning hours of April 4, 2008. The community was connected to the Manitoba electrical grid in 1927, and is the northern terminus of a 260 km, 230 kV electrical transmission line to Harvey, North Dakota.