MicroRNAs (miRNAs) are 19- to 25-nucleotide-long small and noncoding RNAs now well-known for their regulatory roles in gene expression through posttranscriptional and translational controls. Mammalian hibernation is a physiological process involving profound changes in set-points for food consumption, body mass and growth, body temperature, and metabolic rate in which miRNAs may play important regulatory roles. In an initial study, we analyzed miRNAs in the liver of an extreme hibernating species, the Arctic ground squirrel (Spermophilus parryii), using massively parallel Illumina sequencing technology. We identified >200 ground squirrel miRNAs, including 18 novel miRNAs specific to ground squirrel and mir-506 that is fast evolving in the ground squirrel lineage. Comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 mo after hibernation had ended (nonhibernating), we identified differentially expressed miRNAs during hibernation, which are also compared with the results from two other miRNA profiling methods: Agilent miRNA microarray and real-time PCR. Among the most significant miRNAs, miR-320 and miR-378 were significantly underexpressed during both stages of hibernation compared with nonhibernating animals, whereas miR-486 and miR-451 were overexpressed in late torpor but returned in early arousal to the levels similar to those in nonhibernating animals. Analyses of their putative target genes suggest that these miRNAs could play an important role in suppressing tumor progression and cell growth during hibernation. High-throughput sequencing data and microarray data have been submitted to GEO database with accession: GSE19808.