The environmental risk of natural and synthetic ligands of the nuclear progesterone receptor (nPR) has been pointed out, however there is still a lack of mechanistic information regarding their ability to interact with nuclear PR in aquatic species. To identify possible interspecies differences, we assessed in vitro the ability of manifold progestins to transactivate zebrafish (zf) and human (h) PRs, using two established reporter cell lines, U2OS-zfPR and HELN-hPR, respectively. Reference ligands highlighted some differences between the two receptors. The reference human agonist ligands promegestone and progesterone induced luciferase activity in both cell lines in a concentration-dependent manner, whereas the natural zebrafish progestin 17α,20β-dihydroxy-4-pregnen-3-one activated zfPR but not hPR. The potent human PR antagonist mifepristone (RU486) blocked PR-induced luciferase in both cell models but with different potencies. In addition, a set of 22 synthetic progestins were screened on the two cell lines. Interestingly, all of the tested compounds activated hPR in the HELN-hPR cell line, whereas the majority of them acted as zfPR antagonists in U2OS-zfPR. Such zfPR-specific response was further confirmed in zebrafish liver cells. This study provides novel information regarding the activity of a large set of progestins on human and zebrafish PR and highlights major interspecies differences in their activity, which may result in differential effects of progestins between fish and humans.