The biofortification of plants seems to be a beneficial dietary strategy for reducing iron deficiency around the world. The main aim of this study was to determine the content and bioaccessibility of minerals in biofortified lettuce in relation with plant morphological parameters. The experiment was conducted under controlled conditions in a climate chamber. Plants were grown in hydroponics with the application of nutrient solutions with varied iron levels (mg dm^-3): 1.5, 3.0, and 4.5. The contents of Fe, Zn, Cu, Mn, Ni, Mg, Ca, Na, and K in the lettuce were assayed after the dry ashing of the samples. The enzymatic digestion in vitro was performed using the fresh plant, and the contents of released elements were assayed. The concentrations of elements in lettuce before and after digestion were determined by atomic absorption spectrometry. In the lettuce, the contents of total, soluble, and insoluble fiber were also measured. It was found that the highest Fe level significantly increased the yield of dry matter. The studied trends of chlorophyll fluorescense change indicated a positive effect of Fe biofortification on plants but at the same time, an increase in carotenoid content was observed along with an increase in Fe, which may indicate the plants' initial defense response to increasing oxidative stress. Increasing Fe biofortification also caused an increase in chlorophyll content in leaves. The higher content of Fe and most other elements in the plant was in biofortified lettuces. Generally, biofortification of iron decreased Fe bioaccessibility and increased the bioaccessibility of other elements. In conclusion, the content of iron increases, but its bioaccessibility decreases in biofortified lettuce, and plant responses can be affected by biochemical reactions connected among others with carotenoids and chlorophyll content.