Patients with borderline personality disorder (BPD) have a heightened sensitivity to social exclusion. Experimental manipulations have produced inconsistent findings and suggested that baseline negative affect (NA) might influence the experience of exclusion. We administered a standardized social exclusion protocol (Cyberball paradigm) in BPD (n = 39) and age-matched and sex-matched healthy controls (n = 29) to investigate the association of NA on social exclusion and activation in brain regions previously implicated in this paradigm. Compared with controls, patients with BPD showed higher activation during social exclusion in the anterior cingulate cortex (ACC), the medial prefrontal cortex (mPFC), and in the right precuneus. Prescan NA ratings were associated with higher brain activation in the ACC and mPFC over all conditions, and post hoc t tests revealed that differences between the groups were only significant when controlling for NA. Brain activation during exclusion was correlated with NA separately for each group. Only BPD patients showed a significant association of NA and exclusion related precuneus activation (r = .52 p = .001). Additionally, BPD patients experienced less feelings of belonging compared with a healthy control (HC) group during inclusion and exclusion, although they estimated their ball possessions significantly higher than did the HC. These findings suggest that baseline NA has a crucial impact on Cyberball-related brain activation. The results underscore the importance of considering levels of NA in social exclusion protocols for participants high in this trait.