The hypoxia (HPX) acts the brain injury and apoptosis via the Ca2+ influx-induced excessive mitochondria free reactive oxygen species (mitROS) in neurons. The effective treatment of HPX is not possible yet. In addition to the antiviral and antiparkinsonian actions, amantadine (AMN) has been evaluated as a drug in treatments against brain injury. TRPM2 and TRPV4 channels are activated by mitROS. AMN attenuates NMDA receptor-induced Ca2+ influx, mitROS, inflammation, and apoptosis in the brain. However, the molecular pathways underlying AMN's neuroprotection against HPX remain elusive. We investigated the protective role of AMN via attenuation of TRPM2 and TRPV4 on oxidative neurotoxicity, mitochondrial membrane potential (Delta psi m), inflammation, and apoptosis in neuronal cells (SH-SY5Y). The SH-SY5Y and HEK293 cells were divided into six groups as follows: control, AMN (750 mu M for 48 h), HPX (200 mu M CoCl2 for 24 h), HPX + AMN, HPX + TRPM2 blockers (25 mu M ACA or 100 mu M 2APB for 30 min), and HPX + TRPV4 blocker (ruthenium red (RuR)-1 mu M for 30 min). The HPX caused to upregulation of Ca2+ influx with an upregulation of Delta psi m and mitROS. The changes were not observed in the absence of TRPM2 and TRPV4 in the HEK293 cells. When HPX induction, TRPV4 agonist (GSK1016790A) and TRPM2 agonists (ADP-ribose and H2O2)-induced channel activity were diminished by the incubation of AMN and channel antagonists (RuR, ACA, and 2APB). The changes of mitROS, apoptotic markers (caspase-3 and -9), cell death rate, cell viability, cytokine (IL-1 beta, IL-6, and TNF-alpha), Delta psi m, and Zn2+ concentrations were also restored by the incubation of AMN. In conclusion, the treatment of AMN attenuated HPX-mediated mitROS, apoptosis, and TRPM2/TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting the HPX-mediated neurodegenerative and cerebrovascular diseases associated with the upregulation of mitROS, Ca2+, and Zn2+ concentration.