A 14.2 m long core was recovered from the southern Lake Manitoba basin. The sediment, consisting mainly of silty clay, was studied for siliceous microfossil content and mineral magnetics; 14 new accelerator mass spectrometry (AMS) radiocarbon dates provide chronological control of the paleoenvironmental history of the basin. The basal 5 m contains ice-rafted clasts and is largely barren of siliceous microfossils; these sediments were deposited when the lake was part of glacial Lake Agassiz. Sediments immediately overlying the barren part of the sequence contain AMS dates of 7700-7400 BP and reflect a dramatic change in conditions in the basin. Diatom abundances rise abruptly. Magnetic characteristics change substantially. The presence of freshwater taxa such as Stephanodiscus niagarae, together with brackish water diatoms, indicate that shallow, turbid, high-nutrient conditions with variable salinity occurred during the early part of the middle Holocene. Although climatic conditions throughout the northern Great Plains are known to have become drier and warmer during the mid-Holocene, there is a distinct change in diatom taxa in the Lake Manitoba sequence toward less saline conditions at this time. The presence of the riverine diatom Aulacoseira granulata in this interval supports previous conclusions that these freshwater conditions resulted from the northward diversion of the Assiniboine River into the basin. Following this, diatoms indicate an abrupt increase in salinity to >1500 mg·L-1 total dissolved solids between 4000 and 2600 BP, reflecting the diversion of the fresh waters of the Assiniboine River away from Lake Manitoba. Increasingly cooler and wetter conditions during the late Holocene, combined with differential isostatic rebound, caused a freshening of the lake during the late Holocene.