The aim of the present study is to further understand and analyze the interaction of mefloquine with praziquantel against adult Schistosoma japonicum in vitro. Mice infected with S. japonicum cercariae for 35-37 days were sacrificed, and adult schistosomes were collected by perfusion. Schistosomes were placed to each of 12 wells of a Falcon plate and maintained in RPMI 1640 supplemented by 10% calf serum. For determination of 50% and 95% lethal concentration (LC50 and LC95) of the two drugs in vitro, schistosomes were exposed to mefloquine at concentrations of 1, 2, 3, 4, 5, 6, 7, and 10 μg/mL or praziquantel at concentrations of 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 10, and 30 μg/mL. The plate was incubated at 37°C in 95% air + 5% CO₂ for 72 h. According to the half-life of oral mefloquine and praziquantel in mice, mefloquine combined with praziquantel simultaneously, mefloquine administered within 1 h after praziquantel and praziquantel administered within 17 h after mefloquine were used to evaluate the effect of mefloquine in combination with praziquantel against S. japonicum in vitro. The results showed that the LC50 and LC95 of mefloquine calculated by the Bliss method were 6.17 μg/mL (95% confidence limits, 5.84-6.517 μg/mL) and 8.703 μg/mL (95% confidence limits, 7.632-9.797 μg/mL), respectively. As to praziquantel, no worm death was seen when schistosomes were exposed to praziquantel at concentrations of 0.005-0.2 μg/mL for 72 h. While in the worms exposed to praziquantel 1, 10, and 30 μg/mL, strong spasmodic contractions of the worm body and vesiculation along the worm surface were observed, but 48-75% of the schistosomes survived the exposure in 72-h incubation. Meanwhile, the number of dead worms that emerged in each group was not proportion to the increasing concentrations. Therefore, it is not appropriate to calculate the LC50 and LC95 of praziquantel. For evaluation of the interaction with the two drugs, praziquantel 0.1 or 0.2 μg/mL, which may induce moderate or strong spasmodic contractions of the worm body and vesiculation along the worm surface, was combined with mefloquine 5, 6, or 7 μg/mL. It was found that when mefloquine combined with praziquantel simultaneously or administered 1 h after addition of praziquantel, the spasmodic contraction of the male worm body was antagonized by mefloquine in various degrees according to the concentrations of mefloquine used. Meanwhile, praziquantel-induced weakened motor activity could be reversed by mefloquine. In female worms, morphological alterations and stimulated motor activity induced by mefloquine still developed. Interestingly, using these two regimens to combine mefloquine with praziquantel resulted in no impact or a decrease in worm mortality. On the other hand, praziquantel 0.2 μg/mL administered within 17 h after mefloquine 5 or 6 μg/mL promoted the damage to the tegument of the worms, which led to enhance the worm mortality compared with that of worms exposed to mefloquine alone. The results indicate that in vitro higher concentrations of praziquantel administered within 17 h after mefloquine may increase the effect against adult schistosomes, while praziquantel combined with mefloquine simultaneously or administered 1 h before addition of mefloquine exhibits no impact or decrease in the effect against schistosomes.