Fascioliasis is an important zoonotic helminthic disease caused byFasciola hepaticaand poses a serious threat to global public health. To evade the immune response of its host (humans or animals),F. hepaticasecretes various antioxidant enzymes such as glutathione transferase (GST) to facilitate its invasion, migration and parasitismin vivo. To investigate the biological functions of a novel omega-class GST (GSTO), the molecular features of GSTO2 ofF. hepaticawere analyzed by online software, and the biochemical propertiesin vitroof recombinant GSTO2 (rGSTO2) were dissected. Then, the regulatory roles of rGSTO2 protein in murine macrophagesin vitrowere further explored. The results revealed that theGSTO2gene encodes 254 amino acids, which harbor the characteristic N-terminal domain (βαβαββα) and C-terminal domain (α-helical) of the cytoplasmic GST superfamily. GSTO2 was mainly expressed inF. hepaticavitelline follicles, intestinal tract, excretory pores and vitelline cells, with thioltransferase and dehydroascorbate reductase activities. Moreover, rGSTO2 protein could be taken up by murine macrophages and significantly inhibit the viability of macrophages. In addition, rGSTO2 protein could significantly promote apoptosis and modulate the expression of cytokines in macrophages. These findings suggested thatF. hepaticaGSTO2 plays an important role in modulating the physiological functions of macrophages, whereby this protein might be involved in immunomodulatory and anti-inflammatory roles during infection. This study provided new insights into the immune-evasion mechanism ofF. hepaticaand may contribute to the development of a potential anti-inflammatory agent.