Short-term pulsed exposure tests have been increasingly used to evaluate the ecotoxicity of pollutants of which concentrations vary over time in the field. In pulsed exposure, time-weighted average (TWA) concentration is often used as an index of exposure. However, there have been few studies to demonstrate whether TWA concentration can be used to evaluate the effect of endocrine-disrupting chemicals on the daphnids. Pyriproxyfen is one of the juvenile hormone analogs that induces daphnids to produce male offspring. To evaluate whether peak or TWA concentration can explain the effects of pyriproxyfen on daphnid reproduction, we measured the number of offspring and the proportion of male offspring produced by Daphnia magna during 21-day under different exposure treatments, constant, single-pulse, and multi-pulse exposure, at an equivalent TWA concentration. Constant exposure of 50 ng/L pyriproxyfen did not affect either the fecundity or the proportion of male offspring, while a single-pulse exposure of 525 ng/L pyriproxyfen over 2 day at four different age did not reduce fecundity, but the proportion of male offspring increased age dependently. Multi-pulses exposure of 131 ng/L pyriproxyfen over two days four times (total eight days) resulted in a decrease in fecundity and the highest proportion of male offspring. Daily observation demonstrated that male offspring was only produced several days after the exposure to a certain concentration of pyriproxyfen. Therefore, neither TWA nor peak concentration accurately evaluated the effects of pulsed exposure of pyriproxyfen on the reproduction of D. magna, particularly its effect on the proportion of male offspring.