Background: It is well known that circadian rhythms and sleep homeostasis contribute to a pronounced trough in sleepiness and behavioral performance at night. However, the underlying neuroimaging mechanisms remain unclear. How brain-function connectivity is modulated during sleep deprivation (SD) has been rarely examined. Methods: By increasing the number of scanning sessions during SD, the current study used voxel-mirrored homotopic connectivity (VMHC) to investigate dynamic changes in interhemispheric communication during one night of SD. Every 2 hours from 10 pm to 06 am (session 1, 10 pm; session 2, 12 am; session 3, 2 am; session 4, 4 am; session 5, 6 am), functional magnetic resonance-imaging data and Stanford Sleepiness Scale (SSS) scores were collected from 36 healthy participants with intermediate chronotype. Dynamic changes in SSS scores and VMHC were determined using one-way repeated-measure ANOVA with the false discovery-rate method to correct for multiple comparisons. Results: Significant time effects for VMHC were found mainly in the bilateral thalamus, bilateral superior temporal gyrus, and bilateral precentral gyrus. SSS scores and VMHC in these areas were both found to be monotonously increased during SD. Furthermore, significant positive associations were found between SSS valu and VMHC values in the left superior temporal and right superior gyri. Conclusion: These findings might represent the dynamic modulation of circadian rhythm merely or the interaction effects of both circadian rhythm and sleep homeostasis on interhemispheric connectivity within the thalamus, default-mode network, and sensorimotor network. Our study provides more comprehensive information on how SD regulates brain connectivity between hemispheres and adds new evidence of neuroimaging correlates of increased sleepiness after SD.