Constraint-induced movement therapy (CIMT) induces plastic changes of the residual brain after stroke. However, the circuit-level reorganization caused by CIMT and its causal relationship with functional recovery is still unclear. To elucidate these questions, we investigated the role of the reorganized motor circuits in CIMT-treated rat with stroke using double vector-infection technique for pathway-selective blockade. Wistar rats were divided into a sham-operated group, internal capsule hemorrhage (ICH) group, and ICH-CIMT group. ICH was induced by collagenase injection (15 Units/ml, 1.4 µl) into the internal capsule. CIMT group rats wore a one-sleeve cast to restrain their intact forelimb for a week. Behavioral assessments (skilled reaching and ladder stepping) showed better recovery in ICH-CIMT group than in ICH only group. Mapping of the ipsilesional motor cortex revealed that ICH-CIMT group demonstrated larger forelimb representative area compared to ICH group on days 10 and 26. Moreover, microinjection of muscimol (1 µM, 1 µl) into the newly-emerged forelimb areas resulted in impairment of the recovered forelimb movements. Next, we injected biotin dextran amine (BDA) into the same areas to trace axons projecting from these sites. As results, abundant BDA-positive fibers were found around ipsilateral red nucleus. Finally, we used a double-infection technique to block the cortico-rubral pathway selectively (Kinoshita et al., 2012; Sooksawate et al. 2013). A retrograde gene transfer vector with the gene encording enhanced tetanus neurotoxin under the control of a tetracycline responsive element was injected into the red nucleus and subsequent injection of adeno-associated viral vector with a highly efficient Tet-ON sequence at the motor cortex. After 6 weeks, impairment of skilled reaching task of ICH-CIMT group was found when the administration of doxycycline was initiated. These data suggested that the CIMT promoted reorganization of the ipsilesional motor cortex and enhanced the connection of cortico-rubral pathway as a substrate for functional recovery.