【Objective】To explore the regulatory mechanism of miR-204-3p targeting EphB2 gene on proliferation， apoptosis and invasion of NSCLC.【Methods】A549 cells were cultured and divided into 5 groups：NC mimic group，miR- 204-3p mimic group，OE NCgroup，OE EphB2 group，miR-204-3p mimic+ OE EphB2 group. MTT，flow cytometry， Transwell and scratch test were used to detect cell proliferation，cycle，apoptosis，invasion and migration. Double Luciferase Report was used to analyze the targeting relationship between mir-204-3p and EphB2. The mRNA and protein expression of miR-204-3p，EphB2 were detected by qPCR and WB.【Results】Innon-small lung cancer cells，the binding site of mir- 204-3p and EphB2 3′UTR region，the high expression of miR- 204-3p significantly inhibited the expression of EphB2 mRNA and protein（P<0.01）. Compared with the Negtively control group（NC mimic group），the proliferation of A549 cells in miR-204-3p mimicgroup were significantly decrease（P<0.05），and the apototic rate was significantly in⁃ creased（P<0.05）. Also，the cell migration and invasion ability were also decreased significantly（P<0.05）. Transfection of Ephb2 reversed the above changes. In addition，in non-small cell lung cancer tissues，miR-204-3p was negatively cor⁃ related with EphB2 expression （r=0.636，P<0.001），and the overall survival was shorter in EphB2 high expression groups than that in low expression groups（logrank χ2=3.899，P=0.049）.【Conclusion】MiR- 204- 3p  inhibits proliferation，migration and invasion of non-small lung cancer cells and induces apoptosis by down-regulating EphB2.