Ferrous glycinate (Fe-Gly) maintains high bioavailability in animals, but its exact absorption mechanism is still unknown. Here, we studied on the absorption kinetics of ferrous glycinate and its impact on the relevant transport protein in Sprague-Dawley (SD) rats. A total of 72 SD rats (male, BW 100 ± 6.25 g) were randomly allotted to three treatments. These treatments were perfused with 1 mL of normal saline, ferrous sulfate (FeSO4), and ferrous glycinate (71.35 mg/L as iron) separately. Four rats were selected from each treatment for collection of blood from the tails at certain times (15, 30, 45, 60, 75, 90, 120, 240, and 360 min) after gavage. Moreover, other six rats selected from each treatment were slaughtered for sampling after gavage at 2, 4, and 6 h to evaluate the expression of intestinal transport protein. Pharmacokinetic parameters of iron were determined by one-compartmental analysis. Compared with FeSO4, the peak plasma concentration of iron (C max) is higher in the rats given gavage with Fe-Gly (P < 0.05). Four hours after gavage with Fe-Gly, the expression of divalent metal transporter 1 (DMT1) in the duodenum is significantly decreased (P < 0.05), but the expression of ferroportin 1 (Fpn1) is significantly increased (P < 0.05). This study indicates that Fe-Gly as iron sources can be absorbed more and utilized faster than FeSO4, and they had different effects on the expression of intestinal transport protein.