Colonocyte and distal small intestinal enterocyte metabolism was studied in rats fed either an elemental diet or an elemental diet supplemented with 30% mixed dietary fiber for 14 d. Cells were incubated in RPMI 1640 culture media containing 14C-labeled glutamine (1 mmol/L), glucose (11 mmol/L), and short-chain fatty acids: acetate, propionate and butyrate (5 mmol/L). Substrate oxidation and product formation were measured. Colonocytes from the fiber-fed group had 22-51% higher oxidation rates than the elemental-fed group for all substrates tested. The group consuming the fiber diet had 28% less glutamate formation from glutamine by isolated colonocytes than the group consuming the elemental diet. Enterocyte acetate oxidation and lactate formation rates were lower (60 and 30%) in the fiber-fed animals vs. the elemental-fed group. Including short-chain fatty acids and ketone bodies in incubation media differentially affected acetate, glutamine and glucose metabolism in isolated intestinal cells, depending on segment and diet. Short-chain fatty acid and glucose oxidation rates were higher for colonocytes than enterocytes and glutamate formation was greater in enterocytes than colonocytes. Fiber consumption increased this segmental disparity. This study demonstrates that dietary fiber consumption increases substrate oxidation by isolated colonocytes but not distal small intestinal enterocytes.