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Laryngeal squamous cell carcinoma (LSCC), one of the most common malignant solid tumors in the world, has a high rate of mortality, recurrence, and metastasis. Recombinant human arginase (rhArg) recently has been developed in arginine deprivation therapy for a number of malignant tumors. In the present study, we observed that rhArg triggered significant cytotoxicity in human laryngeal squamous cell carcinoma Tu212 cells. Meanwhile, we observed that rhArg simultaneously activated autophagic flux in Tu212 cells, which was demonstrated by the accumulation of autophagosome and light chain 3 II (LC3-II). And, we explored the role of autophagy in cytotoxicity induced by rhArg in Tu212 cells. Autophagy inhibitors including chloroquine (CQ) and bafilomycin A1 (Baf A1) enhanced cytotoxicity induced by rhArg, implying the protective role of autophagy in rhArg-treated Tu212 cells. Moreover, Akt/mTOR signaling pathway was most possibly to participate in the rhArg-induced autophagy. Meanwhile, rhArg could upregulate the phosphorylation of ERK1/2 in a time-dependent manner. Therefore, all the results illuminated the cytoprotective role of autophagy in the treatment of rhArg in laryngeal squamous carcinoma Tu212 cells and provided a potential approach for LSCC therapy by rhArg combined with autophagy inhibitors.