Cancer　cells　can　metabolize　glutamine　to　replenish　TCA　cycle　intermediates　for　cell　survival.　Glutaminase　(GLS1)　is　over-expressed　in　multiple　cancers,　including　colorectal　cancer　(CRC).　However,　the　role　of　GLS1　in　colorectal　cancer　development　has　not　yet　fully　elucidated.　In　this　study,　we　found　that　GLS1　levels　were　significantly　increased　in　CRC　cells.　Knockdown　of　GLS1　by　shRNAs　as　well　as　GLS1　inhibitor　BPTES　decreased　DLD1　and　SW480　cell　proliferation,　colony　formation　and　migration.　Knockdown　of　GLS1　as　well　as　BPTES　induced　reactive　oxygen　species　(ROS)　production,　down-regulation　of　GSH/GSSG　ratio,　an　decrease　in　Nrf2　protein　expression　and　an　increase　in　cytoplasmic　Nrf2　protein　expression　in　DLD1　and　SW480　cells.　Furthermore,　Knockdown　of　GLS1　as　well　as　BPTES　inhibited　autophagy　pathway,　antioxidant　NAC　and　Nrf2　activator　could　reversed　inhibition　of　GLS1-mediated　an　decrease　in　autophagic　flux　in　DLD1　and　SW480　cells.　Depletion　of　GLS1-induced　inhibition　of　DLD1　and　SW480　CRC　cell　proliferation,　colony　formation　and　migration　was　reversed　by　autophagy　inducer　rapamycin.　These　results　suggest　that　targeting　GLS1　might　be　a　new　potential　therapeutic　target　for　the　treatment　of　CRC.