Grey tree frogs (Hyla versicolor) can endure full-body freezing over the winter, with survival aided by metabolic rate depression. Post-transcriptional controls on gene expression include microRNA regulation of gene transcripts that can aid implementation of protein changes required for freezing survival. Western immunoblotting was used to examine protein expression levels of the miRNA biogenesis pathway in three tissues. During freezing, four proteins were upregulated in liver, whereas four proteins in muscle and five proteins in kidney were downregulated. Small RNA-sequencing and bioinformatic analysis of liver showed that seven miRNAs were freeze-upregulated and four were freeze-downregulated. Functions for these miRNAs may involve inhibition of signaling pathways, apoptosis, and nuclear processes. Interestingly, miRNAs may enhance ribosomal biogenesis. Overall, the data show miRNA biosynthesis is altered during freezing and differentially regulated across tissues, with computational predictions highlighting specific functions and processes that may be disproportionally altered during freezing.