In addition to its function of innate immunity against invading pathogens, neutrophil extracellular traps (NETs) promote thrombosis, autoimmune disease, and cancer metastasis; therefore, unnecessary exposure to the triggers of infection-independent NET generation should be avoided. We herein show that inhibition of forward-mode Na+/Ca2+ exchange by amiloride analogs, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N-Methyl-N-isobutyl)amiloride (MIA), triggers NETotic cell death independently of infectious stimuli. Isolated human neutrophils treated with EIPA and MIA undergo NETotic cell death by an increase of intracellular Ca2+ following activation of NADPH oxidase and the resultant upregulation of intracellular ROS. EIPA- and MIA-mediated intracellular Ca2+ increase is attributed to the competitive binding of EIPA and MIA against Na+ to Na+/Ca2+ exchanger 1 (NCX1). These results demonstrate a new mechanism of infection-independent NET generation and implicate NCX1 as a physiologic regulator of intracellular calcium balance and NETotic cell death.