Objective To investigate the effect of doxycycline on the proliferation and vasculogenic mimicry in retinoblastoma (RB) cell line in vitro. Methods RB cell line were tested for their ability to form perfusable tubular networks in 3D culture with doxycycline in the concentrations ranging from 5 to 20 mg/L, and CoCl2 was used as chemical hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. The effect of doxycycline on proliferation were detected by MTT assay in vitro, and the effect on tube formation of RB cells were detected by tube-like structure formation assay and PAS staining. The mRNA levels of MMP2 and MMP9 at different hypoxic culture and different doxycycline concentrations were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Results The micrograph showed that RB cells linked each other to form cavity and network tructure in 3D culture. the number of tubules in doxycycline group were significantly lower than which in the control group in the concentrations ranging from 5 to 20 mg/L (Plt;0.001).OD of doxycycline group was significantly lower than which in the control group (t=15.320,Plt;0.01) , The proliferation of RB cells had a negative correlation with the concentration of doxycycline (r =-0924, Plt;001). The levels of MMP2 and MMP9 mRNA of RB cells under hypoxia were significantly higher than which in the control group (t=16.469,Plt;0.01). As the concentration of doxycycline increased, the expression of MMP-2 and MMP-9 decreased. The result of double staining also showed that VM, formed by CD34negative and PASpositive tumor cells, existed in 12 simples of retinoblastoma. Conclusion RB cells have the capacity of selfmetamorphosing and vasculorizing in 3D culture. Doxycycline can inhibit their proliferation and vasculogenic mimicry formation in vitro by downregulating the expression of MMP-2 and MMP-9 .