In recent years, the safety of copper in drinking water has increasingly been questioned. Copper speciation is an important factor that affects its bioavailability and toxicity; thus, it is critical to investigate the speciation of copper that is ingested from food and drinking water during in vitro digestion. After digestion, water- and food-derived copper formed 60 ± 4% 0.1–1 kDa and 49 ± 6% 10–1,000 kDa copper complexes, respectively. Under simulated fasting drinking water conditions, up to 90 ± 2% 0.1–1 kDa copper complexes formed. In addition, using ion selective electrode analysis, water-derived copper was detected that contained higher Cu²⁺ concentrations after digestion than those of food-derived copper. These results indicate that water-derived copper forms smaller-sized species and exhibits higher Cu²⁺ concentrations during digestion than those of food-derived copper, thereby highlighting the importance of reassessing the safety limit for copper in drinking water.