The objective of this study was to evaluate the interaction of pro‐vitamin A‐rich sweet potato on iron bioavailability of biofortified cowpeas, using in vitro Caco‐2 cells and in vivo depletion–repletion rat model. Mixtures of conventional rice with cultivars of iron‐biofortified (Aracê, Xiquexique, and Tumucumaque) or conventional (Guariba) cowpeas with or without sweet potato biofortified with pro‐vitamin A carotenoids were evaluated. The ratio of ferritin/total protein in Caco‐2 cells was used as the index of cellular Fe uptake in the in vitro assay. The animal study evaluated the hemoglobin gain, the relative biological value, and the gene expression of transferrin and ferritin proteins by reverse transcription polymerase chain reaction. In the in vitro study, Xiquexique cowpea presented higher bioavailability of iron in the absence of sweet potato, and no difference was observed between the other cultivars of cowpea with and without sweet potato. The in vivo bioavailability (relative biological value of hemoglobin regeneration efficiency) differed statistically only between Guariba groups added to sweet potato and Tumucumaque. Ferritin mRNA expression did not differ between the test and control (ferrous sulfate) groups. Regarding the transferrin mRNA expression, there was a difference between the test and control groups except for the Xiquexique group. The association of rice and beans with sweet potato rich in carotenoids favored the gene expression of proteins involved in the iron metabolism, as well as its bioavailability, corroborating beneficial effects of this mixture. Xiquexique cowpea was shown to be the most promising compared to the other cultivars, exhibiting higher iron content in the digestible fraction, better in vitro bioavailability of iron, and transferrin gene expression. Practical Application Data from the study indicated greater in vitro bioavailability of iron for Xiquexique cowpea and sweet potato mixtures, in addition to the greater regeneration efficiency of hemoglobin in vivo as the bioavailability of iron among biofortified beans, highlighting the promising benefits of biofortification.