Objectives: To investigate the differences in functional brain activity and connectivity between nurses working long-term shifts and fixed day shift and explore their correlations with work-related psychological conditions. Methods: Thirty-five nurses working long-term shifts and 35 nurses working fixed day shifts were recruited. After assessing work-related psychological conditions, such as burnout and perceived stress of these two groups of nurses, amplitude of low-frequency fluctuations (ALFF) and functional connectivity (FC) analyses were performed to investigate the between-group differences in brain functional activity and connectivity. Furthermore, correlation analysis between the ALFF/FC metrics and psychological conditions was conducted. Results: Compared with nurses working fixed day shifts, nurses working long-term shifts showed higher levels of burnout, perceived stress, and depression scores; lower z-transformed ALFF (zALFF) values in the right dorsolateral prefrontal cortex (dlPFC), right superior parietal lobule (SPL), and right anterior cingulate cortex (ACC); and higher zALFF values in the right middle temporal gyrus (voxel-level p 0.001, cluster-level p 0.05, gaussian random field (GRF) correction), as well as lower FC values in the right dlPFC-right SPL and right dlPFC-right ACC (p 0.05, false discovery rate (FDR) corrected). Moreover, the FC values in the right dlPFC-right SPL were negatively correlated with the perceived stress score in nurses working long-term shifts (p 0.05, FDR corrected). Conclusions: This study demonstrated that nurses working long-term shifts had lower functional activity and weaker functional connectivity in the right frontoparietal network, which mainly includes the right dlPFC and right SPL, than those working on regular day shift. The current findings provide new insights into the impacts of long-term shift work on nurses’ mental health from a functional neuroimaging perspective.