AbstractIn motor learning, sequence specificity, i.e. the learning of specific sequential associations, has predominantly been studied using task-based fMRI paradigms. However, offline changes in resting state functional connectivity after sequence-specific motor learning are less well understood. Previous research has established that plastic changes following motor learning can be divided into stages including fast learning, slow learning and retention. A description of how resting state functional connectivity after sequence-specific motor sequence learning (MSL) develops across these stages is missing. This study aimed to identify plastic alterations in whole-brain functional connectivity after learning a complex motor sequence by contrasting an active group who learned a complex sequence with a control group who performed a control task matched for motor execution. Resting state fMRI and behavioural performance were collected in both groups over the course of 5 consecutive training days and at follow-up after 12 days to encompass fast learning, slow learning, overall learning and retention. Between-group interaction analyses showed sequence-specific decreases in functional connectivity during overall learning in the right supplementary motor area (SMA). We found that connectivity changes in a key region of the motor network, the superior parietal cortex (SPC) were not a result of sequence-specific learning but were instead linked to motor execution. Our study confirms the sequence-specific role of SMA that has previously been identified in online task-based learning studies, and extends it to resting state network changes after sequence-specific MSL.