Carcinoma-associated fibroblasts (CAFs) are a key determinant in malignant progression of cancer and represent an important target for cancer therapies. In this work, we present a microfluidic-based 3D co-culture device to reconstruct an in vitro tumor microenvironment and firstly investigate the effect of CAFs on cancer cell invasion in 3D matrix. This device is composed of six co-culture units, which enable parallel co-culture assays to be run in the presence of 3D extracellular matrix. Salivary gland adenoid cystic carcinoma (ACC) cells and CAFs embedded in matrix were co-cultured without direct contact on the device. Communication between ACC cells and CAFs could be established via medium diffused in matrix. It was observed that CAFs promoted ACC cell invasion in 3D matrix in a spheroid fashion, indicating that CAFs play a critical role in cancer invasion. We further demonstrated the effect of MMP inhibitor as an agent against CAF-promoted cancer invasion. This co-culture device reproducibly reflected the in vivo growth and invasion pattern of ACC and recreated the stroma-regulated ACC invasion. Thus, it provides a suitable platform for elucidating the mechanism of CAF-regulated cancer invasion and discovering anti-invasion drugs in a well defined 3D environment.