Yogurt acid whey (YAW) contains significant amounts of calcium as well as small amounts of protein, thus the idea of its reintroduction, especially of its calcium content, to the food chain is attractive. Calcium in milk is mainly complexed with casein micelles, whereas YAW contains only small amounts of protein, with no caseins at all, differing substantially from milk in the form in which calcium occurs. Therefore, the objective of the present research paper was to evaluate whether calcium bioavailability differs between YAW and milk. Following the INFOGEST protocol for simulated digestion and by coupling it with the Caco-2 model for intestinal absorption, calcium in YAW had higher bioaccessibility than calcium in milk. However, there were no differences in calcium transport by the intestinal cells and the transcription level of calcium absorption-related genes (VDR, TRPV6, S100G and PMCA1). Lastly, there were no differences in calcium bioaccessibility and the transcription of the calcium absorption-related genes between YAW samples of bovine, ovine or caprine origin obtained from Greek dairy products enterprises. In conclusion, despite the major differences in the protein profile between YAW and milk, there were no differences in calcium transport by the cells, but YAW was associated with higher calcium bioaccessibility, which ultimately may result in higher amount of absorbed calcium.