Fibroblasts and tumor cells have been involved in the process of cancer development, progression and therapy. Here, we present a simple microfluidic device which enables to study the interaction between fibroblasts and tumor cells by indirect contact co-culture. The device is composed of multiple cell culture chambers which are connected by a parallel of cell migration regions, and it enables to realize different types of cells to communicate each other on the single device. In this work, human embryonic lung fibroblasts cells were observed to exhibit obvious migration towards tumor cells instead of normal epithelial cells on the co-culture device. Moreover, transdifferentiation of human embryonic lung fibroblast cells was recognized by the specific expression of a-smooth musle actin, indicating the effect of tumor cells on the behavior of fibroblasts. Furthermore, multiple types of cell co-culture can be demonstrated on the single device which enables to mimic the complicated microenviroment in vivo. The device is simple and easy to operate, which enables to realize real-time observation of cell migration after external stimulus. This microfluidic device allows for the characterization of various cellular events on a single device sequentially, faciliating the better understanding of interaction between heterotypic cells in a more complex microenvironment.