Objective To cultivate C2C12 cells on the modified moldgrooves of Sylgard 184 and induce C2C12 cells differentiating into highly organized threedimensional muscle tissue. Methods Bicomponents Sylgard 184 was mixed uniformly by the ratio of 10 ∶1 and poured into 6-well plates.Solidified by standing at room temperature, the surface of Sylgard 184 moldgrooves was casted and washed with Hank solution. Matrigel matrix and collagen were mixed uniformly, then paved at the bottom of grooves and airdried in the biological safety cabinet , making sure that the cell matrix material of coverage was uniform. After UV disinfection, C2C12 cells suspension was inoculated. When cells reached to 80% confluence,then C2C12 cells were differentiation cultivated. The morphology of myotubes was observed under the inverted microscopy. RT-PCR was used to test expression of myogenin and desmin mRNA in skeletal muscle tissue. Expression of myogenin and desmin muscle-specific protein was tested by immunofluorescence.Morphologies and inter-connections of myotubes were observed under scanning electronic microscope. Results As C2C12 cells were differentiation cultivated in the mold-grooves of Sylgard 184 for 7 days, differentiation polarity and myotube fusion were observed under the inverted microscope. After 21 days, it showed that myotubes stood closely and connected with each other detected by the scanning electron microscopy.The thickness of the membrane-like structure with the character of three-dimensional was 0.15mm.The positive expressions of myogenin and desmin mRNAs were detected in skeletal muscle tissue by RT-PCR. Immunofluorescence and DAPI nuclear staining showed that myogenin and desmin gene protein were expressed positively. Conclusion The modified mold-grooves of Sylgard 184 is able to guide the differentiation of C2C12 cells and promotes them to form parallel multinucleated myotubes . Myotubes could overlap with each other and form three-dimen