In this research, our aim was to isolate and characterize the substance known as “meat factor,” which is reported to enhance nonheme iron absorption. We used various analytical techniques, and the final step was a human study to measure the effect of a candidate compound on iron absorption. Lean beef was selected for study, as it is known to increase nonheme iron absorption. Cooked ground beef was homogenized and aliquots were taken through a simulated gastric and intestinal digestion. This was followed by purification using fast protein liquid chromatography. The fractions were collected and applied to a Caco-2 cell system designed to measure iron absorption using radioiron. Fractions with an enhancing effect were analyzed by mass spectrometry, nuclear magnetic resonance, and HPLC, and a proposed empirical formula was obtained for the substance in the most active fraction (C8H20 NO6P). Tandem mass spectrometry was used to identify the compound as L-α-glycerophosphocholine (L-α) by comparing the spectra against authentic material. We added a commercially available food grade source of L-α to vegetarian lasagna, with and without 100 mg ascorbic acid (a known enhancer of nonheme iron absorption), at the same enhancer:iron molar ratio (2:1), and fed meals to 13 women of child-bearing age with low iron stores. The nonheme iron was labeled with stable isotopes of iron to provide a total dose per meal of 10 mg iron, and absorption was measured from erythrocyte incorporation. Nonheme iron absorption from lasagna was increased by the addition of either ascorbic acid (P = 0.010) or L-α (P = 0.023). We have identified L-α as a component of muscle tissue that enhances nonheme iron absorption, and this finding provides new opportunities for iron fortification of foods.