AbstractDeficient human diet is usually reduced in many nutrients, but animal studies on iron absorption have been only carried out for rats fed well-balanced (control) and iron-deficient diets. The aim of this study was to evaluate the effect of iron or iron/zinc supplementation on iron apparent absorption (IAA) in rats fed a diet reduced in all vitamins and minerals (R). The study was conducted on 77, 6-week-old male Wistar rats in 3 stages as follows: stage I, 4-week period of adaptation to R diet (50% less vitamins and minerals compared to AIN-93M recommendations); stage II, 4-week supplementation period with iron (RSFe) or iron/zinc (RSFeZn); stage III, 2-week post-supplementation period (rats fed R diet). Feces samples to IAA determination were collected at the 20–22nd days of stage I and II and the 10–12th days of stage III. To determine the changes in IAA after introducing and discontinuation of supplementation, feces were collected for 3 days after introducing (stage II) and 5 days after the supplementation discontinuation (stage III). At the end of stage II, compared to R rats, the IAA was statistically significantly higher in RSFe and RSFeZn rats (30.3 ± 2.0% vs. 47.4 ± 1.2% and 51.0 ± 1.7%, respectively). After introducing iron or iron/zinc supplementation, the IAA stabilized fast already in the first day, while after discontinuation of the supplementation, at least 4 days was required to the stabilization. At the end of stage III, the IAA was significantly lower in RSFe (15.8 ± 6.6%) than in RSFeZn rats (43.4 ± 5.9%). In conclusion, to confirm that iron and zinc supplementation is more beneficial than iron supplementation only, especially after discontinuation of supplementation, further research among humans is necessary.