This paper investigated the effect of extracellular matrix (ECM) on the morphology and activation status of astrocyte in microfluidic cell culture chip. The device provides a 3D co-culture system mimicking in vivo environment of central nervous system as well as axon isolation from neuron soma. Astrocyteswere seeded in collagen gel through small fluidic channels and neuron cells were aggregated at openings of small channel to large channels. This method not only separated neuronal soma from axon but also segregatedneuronalbody from astrocyte. The interaction of axons and astrocyte in the chip showed that the properties of extracellular matrix determines the activation status of astrocytes.The morphology of astrocytes was compared in two extracellular matrix such as collagen, a mixture of collagen andlaminin. The shape of astrocytes in laminin mixture was more star-like that was almost same as in-vivo astrocyte than that in collagen only.Also the effect of TGF-β1 was higher in laminin mixture, which imply that the astrocytes in laminin mixture were deactivated and those in collagen appeared quiescent. Theexperimental results showed thatthe activation status of astrocytes can be controlled in 3D cell culture chip by the control of extracellular matrix material and the treatment of TGF-β1. Hence the suggested co-culture chip is expected to provide a model of axon growth after the damageof nervous system like spinal cord injury.