Objectives Acute sleep deprivation (SD) seriously affects cognitive functions, such as attention, memory, and response inhibition. Previous neuroimaging studies have demonstrated a close relationship between the functional activities of the precuneus (PC) and the function of alert attention. However, the specific effect of the PC on attention decline after acute SD has not been elucidated. In this study, we used resting-state functional magnetic resonance imaging (fMRI) to study the relationship between the changes of the PC functional connectivity and alertness decline after total SD. Methods Thirty healthy, right-handed adult men participated in the experiment. Alert attention and functional connectivity were assessed by the Psychomotor Vigilance Test and a resting-state fMRI scan before and after total SD. The region of interest to region of interest ("ROI-to-ROI") correlation was employed to analyze the relationship between the PC and other brain regions after acute SD. Results Participants showed decreased alert attention after total SD. In addition, SD induced decreased functional connectivity between the right PC and the right middle frontal gyrus (MFG). Moreover, there was a significant correlation between the decreased PC functional connectivity and alertness decline after total SD. Conclusion Our findings suggest that the interruption of the connection between the right PC and the right MFG is related to the observed decline in alert attention after acute SD. These results provide evidence further elucidating the cognitive impairment model of SD.