© 2019 Elsevier Ltd Papain is an enzyme widely studied due to its multiple applications in foods, brewery, and the pharmaceutical industry, among others. Food-grade papain was treated with pulsed electric field technology to evaluate possible changes on its residual activity (RA), relative protein concentration (RPC), sulfhydryl group content, and structural changes. Three factors were evaluated: electric field (10 and 13 kV/cm), number of pulses (48–432), and flow rate (0.2, 0.4 and 0.6 L/min). Maximal inactivation and decrease in SH groups of papain (64 and 20% respectively) were obtained when a PEF process with 13 kV/cm, 288 pulses and 0.2 L/min was used. The maximal decrease in RPC (18%) was attained when 10 kV/cm, 0.4 L/min, and 144 pulses were applied on the enzyme. Important changes in papain secondary and tertiary structures as a result of the PEF treatments were noticed. PEF technology can be considered then as an effective process to reduce the activity of papain.