Myelin disorders cause cognitive dysfunction, but little is known about how abnormal myelin sheath affects neural activities at the network level. One reason for the lack is a technical difficulty in simultaneous monitoring of changes in both the axonal conduction and network activity. Then, we aimed to develop a culture device to detect myelination dependent changes in axonal conduction velocity in a neuronal network. The photolithographically fabricated device has microtunnels for guiding axons. Two microelectrodes and an oligodendrocyte (OL) culture compartment are set at each microtunnel. This configuration allows us to monitor changes in conduction velocity of axons wrapped by OLs. Neurons and OLs dissected from rat cortical tissues were cultured in the culture device. An immunocytochemical study indicated axonal growth and maturation of OL at 42 days in vitro (DIV), suggesting that neuron-OL co-culture was maintained in microtunnels. Propagating action potentials of individual axons were detected from spontaneous neural activities with a spike sorting method and their conduction velocities were examined. Conduction velocity without seeding OLs was 0.31 m/s, which was consistent with that of previous reports with unmyelinated axons. Although no apparent myelin sheath was observed in OL culture compartments, conduction delay with seeding OLs was approximately half as long as that without seeding OLs at 45 DIV. These results suggest that the culture device enables us to detect the OL-regulated changes in axonal conduction in the neuronal network.