Total sleep deprivation (TSD) induces aberrant insula functional connectivity (FC). The insula comprises at least three functionally distinct subregions: anterior dorsal (dAI), anterior ventral (vAI), and posterior insula (PI). Biased attention toward the anterior insula (AI) has limited our complete understanding of the TSD impact. We aimed to investigate TSD-induced functional connectivity and lateralization across the insula subregions. A total of 54 healthy young men completed 36-h TSD. Two sessions of psychomotor vigilance task (PVT) and 3T resting-state functional magnetic resonance imaging (rs-fMRI) scanning were carried out. A seed-based FC was conducted using bilateral insula subregions. Impaired vigilance, altered functional connectivity and lateralization were observed after TSD. The AI showed enhanced connectivity with the cerebellum, middle frontal gyrus, putamen, and postcentral gyrus but decreased connectivity with the temporal lobes, angular gyrus, calcarine sulcus, anterior cingulum, and medial orbitofrontal gyrus. The PI had increased connectivity with the middle frontal, inferior temporal, and inferior parietal gyrus but showed an anti-correlation with the middle temporal regions, posterior cingulum, and angular gyrus. All the seeds showed ipsilateral connections with specific brain regions, excluding the cerebellum. Both the vAI and PI displayed FC with the insula. The correlation analysis between PVT and brain signal changes did not survive Bonferroni correction. This study provided information about potential functional asymmetries of insula subregions caused by 36-h TSD. These findings provided new insights into the neural mechanisms of inter-hemispheric communication and coordination, which is essential for understanding the overall brain function. Keywords: rs-fMRI, insular subregions, PVT, sleep deprivation, functional connectivity