Tin whiskers grown on a Sn/Cu-plated Polyimide (PI)-flexible substrate are a serious problem in electronic industrial application, because the whiskers lead to catastrophic electrical short circuit failures. Here, we report characterization of microstructures in the whiskers grown on a surface of Sn/Cu-plated PI-flexible substrate by crosssectional transmission electron microscopy (TEM) to analyze the behavior and the formation mechanism of whiskers. The whisker was found to be monocrystalline beta-Sn and grown with the preferred directions of [110] and [101]. The whiskers formed on this tin surface are nucleated and grown by the compressive stress, that is induced externally by insertion of Sn/Cu-plated PI-flexible substrate into the connector.