Stacked crop rotations and improved cultural practices have been used to control pests, but their impact on soil total carbon (STC) (soil organic carbon [SOC] + soil inorganic carbon [SIC]) and crop yield are lacking. We evaluated the effects of stacked vs. alternate-year rotations and cultural practices on STC at the 0- to 125-cm depth and annualized crop yields from 2005 to 2011 in the northern Great Plains. Stacked rotations were durum (Triticum turgidum L.)–durum–canola (Brassica napus L.)–pea (Pisum sativum L.) (D–D–C–P) and durum–durum–flax (Linum usitatissimum L.)–pea (D–D–F–P). Alternate-year rotations were durum–canola–durum–pea (D–C–D–P) and durum–flax–durum–pea (D–F–D–P). A continuous durum (CD) was used as a reference. Cultural practices were traditional (conventional till, recommended seed rate, broadcast N fertilization, and reduced stubble height) and ecological (no-till, increased seed rate, banded N fertilization, and increased stubble height) treatments. Annualized crop biomass residue returned to the soil and grain yield were greater with D–C–D–P and D–D–C–P than D–D–F–P and greater with the ecological than the traditional practice. The STC concentration increased with depth and was greater with CD and D–C–D–P than D–D–C–P and D–D–F–P in traditional and ecological practices at 20 to 50 cm. At 50 to 88 cm, STC concentration was greater with D–F–D–P than D–D–F–P in the traditional practice. At 0 to 125 cm, STC content was lower with D-D-F-P than other crop rotations. Stacked rotations, especially D–D–F–P, reduced soil C storage and crop yields compared with alternate-year rotations. For enhancing soil C storage and crop yields, alternate-year crop rotations are recommended.