In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.