The nonsteroidal ecdysone mimic 1,2-dibenzoyl-1-tert-butylhydrazine (RH-5849), a novel insect growth regulator, is mainly registered for use in rice fields. So far, its toxicity and ecological risks towards aquatic cladoceran invertebrates remain unclear. In this study, RH-5849 was evaluated for its acute and 21-day chronic toxicity towards Daphnia magna. The viability, morphology, growth, and reproduction of D. magna were observed to establish a concentration-toxicity relationship associated with the RH-5849 exposure. In addition, the relationship between the changes of physiological and biochemical indices and the chronic indices was analyzed in order to find potential early warning indicators in D. magna to the chronic risk of RH-5849 exposure. The results showed that the 48-h EC₅₀ of acute immobilization and EC₅₀ of 21-day survival of RH-5849 on D. magna were 45.3 and 1.34 mg/L, respectively. Chronic exposure to RH-5849 mainly affected the reproductive parameters of D. magna and the no observed effect concentration (NOEC) and the EC₅₀ were 0.050 and 0.5423 mg/L, respectively. The number of offspring per female reduced significantly after 21-day exposure to 0.10 mg/L of RH-5849. The morphological changes, manifested in head width and body length, the length of the helmet or apical spine, and the curvature and transparency of the body, were observed in RH-5849-treated groups. Moreover, it was found that the alkaline phosphatase activity in D. magna after 5–7-day exposure was positively correlated with the number of offspring per female after 21 days. These results indicate the potential risk of RH-5849 towards aquatic crustaceans should be taken into consideration when applied to rice fields.