Most fishes with commercial importance from the São Francisco basin are migratory and do not complete the reproductive cycle in lentic environments, such as hydroelectric plant reservoirs, hence natural stocks are declining and there is an urgent need to reduce the pressure of fishing on those wild populations. Therefore, studies on reproductive biology and its relationship with endocrine and environmental factors are key to improving the cultivation techniques of Brazilian fish species. This study examined the influence of water temperature on sex steroid concentrations (testosterone, 17β-estradiol and 17α-hydroxyprogesterone), spawning efficiency, fecundity, fertilisation rate, larval abnormality rates and involvement of the cytoskeleton during the final oocyte maturation of Prochilodus argenteus under experimental conditions. The results of our study showed that in captivity, sex steroid plasma concentrations and spawning performance of P. argenteus were clearly different for fish kept in water with different temperature regimes. In lower water temperature (23°C), it was observed that: 33% of females did not ovulate, fecundity was lower and vitellogenic oocytes after the spawning induction procedure exhibited a smaller diameter. Moreover, concentrations of 17β-estradiol and 17α-hydroxyprogesterone were lower and there was a delay in the final oocyte maturation and, consequently, ovulation and spawning. Our experiments showed direct influence of water temperature in the process of induced spawning of P. argenteus. Changes in water temperature also suggest the tubulin involvement in the nuclear dislocation process and the possible action of actin filaments in the release of polar bodies during final oocyte maturation of P. argenteus.