Praziquantel (PZQ) is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ), which is composed of ( R)-PZQ and ( S)-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers ( R)-PZQ and ( S)-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that ( R)-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2). However, in contrast to ( R)-PZQ, the ( S)-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, ( R)-PZQ showed lower cytotoxicity on SH-SY5Y cells than ( S)-PZQ. Meanwhile, ( R)-PZQ at <80 μM concentration could promote proliferation of macrophage cells (Raw264.7). Our research revealed that ( R)-PZQ has lower cytotoxicity than ( S)-PZQ and has similar cytotoxicity with rac-PZQ. ( S)-PZQ is the principal enantiomer to cause side effects on human definitive hosts. These findings gave the reasonable reasons for World Health Organization to produce ( R)-PZQ as a replacement for rac-PZQ for the treatment of schistosomiasis.