To find out the optimal treatment sessions of repetitive transcranial magnetic stimulation (TMS) over the primary motor cortex (M1) for upper extremity dysfunction after stroke during the 6-week treatment and to explore its mechanism using motor-evoked potentials (MEPs) and resting-state functional magnetic resonance imaging (rs-fMRI), 72 participants with upper extremity motor dysfunction after ischemic stroke were randomly divided into the control group, 10-session, 20-session, and 30-session rTMS groups. Low-frequency (1 Hz) rTMS over the contralesional M1 was applied in all rTMS groups. The motor function of the upper extremity was assessed before and after treatment. In addition, MEPs and rs-fMRI data were analyzed to detect its effect on brain reorganization. After 6 weeks of treatment, there were significant differences in the Fugl-Meyer Assessment of the upper extremity and the Wolf Motor Function Test scores between the 10-session group and the 30-session group and between the 20- and 30-session groups and the control group, while there was no significant difference between the 20-session group and the 30-session group. Meanwhile, no significant difference was found between the 10-session group and the control group. The 20-session group of rTMS decreased the excitability of the contralesional corticospinal tract represented by the amplitudes of MEPs and enhanced the functional connectivity of the ipsilesional M1 or premotor cortex with the the precentral gyrus, postcentral gyrus, and cingulate gyrus, etc. In conclusion, the 20-session of rTMS protocol is the optimal treatment sessions of TMS for upper extremity dysfunction after stroke during the 6-week treatment. The potential mechanism is related to its influence on the excitability of the corticospinal tract and the remodeling of corticomotor functional networks.