The sperm-specific Ca2+ channel CatSper (cation channel of sperm) is vital for male fertility. Contradictory findings have been published on the regulation of human CatSper by the endogenous steroids estradiol, testosterone and hydrocortisone, as well as the plant triterpenoids lupeol and pristimerin. This aim of this study was to elucidate this controversy by investigating the action of these steroids and plant triterpenoids on human CatSper using population based Ca2+-fluorimetric measurements, the specific CatSper inhibitor RU1968, and a functional test assessing the CatSper-dependent penetration of human sperm cells into methylcellulose. Estradiol, testosterone and hydrocortisone were found to induce Ca2+-signals in human sperm cells with EC50 values in the lower μM range. By employing the specific CatSper inhibitor RU1968, all three steroids were shown to induce Ca2+-signals through an action on CatSper, similar to progesterone. The steroids were found to dose-dependently inhibit subsequent progesterone-induced Ca2+-signals with IC50 values in the lower μM range. Additionally, the three steroids were found to significantly increase the penetration of human sperm cells into methylcellulose, similar to the effect of progesterone. The two plant triterpenoids, lupeol and pristimerin, were unable to inhibit progesterone-induced Ca2+-signals, whereas the CatSper inhibitor RU1968 strongly inhibited progesterone-induced Ca2+-signals. In conclusion, this study supports the claim that the steroids estradiol, testosterone and hydrocortisone act agonistically on CatSper in human sperm cells, thereby mimicking the effect of progesterone, and that lupeol and pristimerin do not act as inhibitors of human CatSper.