Fort Assiniboine is a hamlet in northwest Alberta, Canada, within Woodlands County. It is located along the north shore of the Athabasca River at the junction of Highway 33 and Highway 661. It is approximately 39 kilometres (24 mi) northwest of Barrhead, 62 kilometres (39 mi) southeast of Swan Hills and 91 kilometres (57 mi) northeast of Whitecourt.

Fort Assiniboine was founded as a trading post by the Hudson's Bay Company and became a stopping point along the Klondike Trail. It gets its name from the Assiniboine people. The fort itself no longer exists, but the land on which it stood is designated as a National Historic Site for its archaeological value. The hamlet, built on and around the site of the fort, is now a local hub for the surrounding agricultural region.

Local oral history tells of an early (possibly late 1700s) North West Company fur trading post south of Holmes Crossing (an early ferry crossing) on the Athabasca River. In 1821, the North West Company was merged with Hudson's Bay Company (HBC), who then undertook to reorganize its transportation routes, seeking out advantages and efficiencies in its operations. By then, trade on Lesser Slave Lake was in decline and the council adopted a resolution in 1823 calling for a fort further up the Athabasca River to reduce transport times. Under the new plan, Fort Assiniboine on the Athabasca River (the fort was originally named 'Athabaska River House') opened in 1824.

It became the northwest end of an overland 130-kilometre (80 mi) horse track to Edmonton House/Fort Edmonton, cut by Jacques Cardinal, a Métis free trader, in 1824–25. The trail became known as 'The Hudson's Bay Packtrail'.

The trail shortened the distance from Jasper House and the Athabasca Pass within the Rocky Mountains, to Fort Edmonton and thence to York Factory on the Hudson Bay. A party on horseback could make the trip from Edmonton to Fort Assiniboine in two to six days, depending on conditions.

The new route was used by the York Factory Express. The old canoe route involved going far north-northeast down the Athabasca to Fort Chipewyan and then southeast through Methye Portage to Lake Winnipeg.

Though the fort never grew as large as some other Alberta forts, its role as a transportation hub and provision centre ensured its survival between the 1820s and 1880s. The fort burned to the ground after its abandonment by the HBC. No plans existed for its original layout, but, using clues from post administrators' log books and archaeological surveys, a replica was built on the site in 1980. It operates as a museum and friendship centre.

In 1898, when the Chalmers, or Klondike Trail was cut through the Swan Hills, northwest of Fort Assiniboine to Lesser Slave Lake, the location again became a stopping point, with gold seekers crossing the river with a self-service ferry on their overland trek to the Yukon.