The copepod Apocyclops royi tolerates very low salinity while being highly capable of synthesizing n‐3 fatty acids based on PUFA‐poor diets. The feasibility of culturing A. royi in freshwater fed with baker's yeast was evaluated. The copepods were adjusted for 8 days by decreasing stepwise from salinity 20 to freshwater diluting with deionized water. Initially, the cultures exhibited adequate survival and reproduction. However, after 10 days in freshwater, they stopped reproducing presumably due to the lack of vital ions in the medium. Therefore, before continuing the freshwater cultures, we tested the survival of a batch of salinity 1 pre‐acclimated adults and copepodites in various types of freshwater: deionized, bottled ‘mineral’ and tap water for 48 h. All copepods died in deionized water, but a significantly higher survival, 29% ± 11%, was found in mineral water versus 11% ± 8% in tap water. Hence, we continued culturing the copepods in freshwater using ‘Maglekilde spring’ water as diluter. The copepods regained and kept reproducing until day 71 where the experiment was terminated. After a 48 h freshwater versus salinity 20 exposure, nauplii survival was significantly less in freshwater 25 ± 12 versus 79 ± 20 (±STD) at salinity 20 but not different in mean body length. For the first time, we conducted NMR metabolomics revealing a large decrease in glycine betaine and a large increase in lactate in the copepods when challenged by freshwater versus salinity 20. There is obviously a ‘price to pay’ when culturing not only the copepods in freshwater, but also perspectives in developing a ‘universal’ A. royi live‐feed product.