Although 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) and 17α, 20β, 21-trihydroxy-4-pregnen-3-one (20β-S) have been identified as maturation-inducing steroids (MIS) in several teleosts, to date, no MISs have been identified in sturgeons. As it remains possible that an unidentified steroid is an MIS in sturgeons, this study aimed to identify a sturgeon MIS via comprehensive analyses and maturation-inducing (MI) assay of C21 steroids. In vivo and in vitro comprehensive analyses of C21 steroids revealed that serum DHP concentrations were rapidly elevated in the oocyte maturation phase and the DHP production level was notably high among C21 steroids. MI assay indicated that the MI activity of DHP, 17α-hydroxyprogesterone (17OHP), a precursor of DHP, 17α, 20α-dihydroxy-4-pregnen-3-one (αDHP), and 20β-S was high among C21 steroids, but the MI activity of these steroids were similar. In the C21 steroids produced in ovarian follicles during oocyte maturation, 17OHP, αDHP, and unidentified compounds had a low production level, and 20β-S was suggested to be metabolized from DHP after oocyte maturation. Against this background, this study concluded that DHP is a steroid that possesses strong MI activity and is highly produced during oocyte maturation. Although this study could not identify an MIS in sturgeons by fractionation of plasma and subsequent bio assay, it was suggested that DHP is a major MIS in sturgeons.