Calcium is an essential nutrient for human health, because it is a structural component and takes part in a variety of biological processes. The aim of this study was to increase Ca content of baby leaf vegetables (BLV: basil, mizuna, tatsoi and endive), as fresh-cut products. For the production of biofortified BLV, a floating system with two level of Ca (100 and 200 mg L−1) in the nutrient solution was used. In addition, the assessment of bioaccessibility of Ca, by in vitro digestion process, was performed. In all vegetables, the Ca biofortification (200 mg L−1) caused a significant Ca enrichment (9.5% on average) without affecting vegetables growth, oxalate contents and marketable quality. Calcium bioaccessibility ranged from 25% (basil) to 40% (endive) but the biofortified vegetables showed more bioaccessible Ca. These results underline the possibility to obtain Ca biofortified BLV by using agronomic approaches.