Objective To explore the effects of mental fatigue on attention maintenance function by electroencephalogram (EEG) signal characteristics and cortical source analysis. Methods A total of 25 healthy males were recruited as subjects and contingent negative variation (CNV) auditory paradigm was used to assess the differences in EEG characteristics before and after mental fatigue, with the average amplitude of CNV at different processing stages as the analysis indices. Then, the 3-dimensional distribution of cortical current density changes of CNV after mental fatigue were calculated by standardized low-resolution electromagnetic tomography analysis (sLORETA). Results The reaction time of the CNV signal remained unchanged following mental fatigue (P＞0.05), while the lapse rate exhibited a significant increase (P＜0.05). Besides, mental fatigue was related to a notable decrease in the amplitude of CNV early components (500-1 000 ms after warning stimulus) at the central and central parietal electrodes, and a significant reduction in the amplitude of CNV late components (2 550-3 050 ms after warning stimulus) at the prefrontal, frontal, central, and central parietal electrodes (all P＜0.05). The results of sLORETA source analysis showed that the brain activity in the left posterior insular cortex decreased after mental fatigue during the late component of CNV (P＜0.05). Conclusion The decreased activation of the posterior insula, which plays a crucial role in sensorimotor information integration, could potentially serve as a neural mechanism for the reduction of CNV amplitude and the impairment of attention maintenance function following mental fatigue.