The stability of the interface between two liquids in T-shape confluence of microchannels was studied experimentally and numerically. The width and depth of the channels examined in this study were 400 mum and 150 mum, respectively. Visualization showed that fluctuation of the interface occurred in the Re range of above 25. Further, it was observed that one liquid penetrated the other through a symmetrical plane at Re of around 70. On the other hand, numerical simulation showed that the length of the adverse pressure gradient region near the corners increased with Re monotonously. It was confirmed that the slope apparently become smaller at Re = 25. Further, it was observed that a twisted flow was formed near this region, and this intensity increased with Reynolds number. Based on these results, it was supposed that the interaction of two twisted flows near the corners caused the fluctuation of the interface. Further, it was shown numerically that a three-dimensional vortex appeared near the interface formed near the wall at Re of 69. Since the shape of the vortex is very similar with that of the observed penetration, it was concluded in this study that the vortex is the main factor that causes the penetration.