During liver regeneration, normally quiescent liver cells reenter the cell cycle, nonparenchymal and parenchymal cells divide, and proper liver architecture is restored. The gene expression programs regulating these transitions are not completely understood. MicroRNAs are a newly discovered class of small regulatory RNAs that silence messenger RNAs by binding to their 3′-untranslated regions (UTRs). A number of microRNAs, including miR-21, have been shown to be involved in regulation of cell proliferation. We performed partial hepatectomies on mice and allowed the liver to regenerate for 1, 6, 12, 24, and 48 h and 4 and 7 days. We compared the expression of miR-21 in the posthepatectomy liver to the prehepatectomy liver by Northern blot and found that miR-21 was upregulated during the early stages of liver regeneration. NF-κB signaling is also activated very early during liver regeneration. It has been previously reported that NF-κB upregulates the miR-21 precursor transcript. The predicted miR-21 target, Pellino (Peli1), is a ubiquitin ligase involved in activating NF-κB signaling. We observed an inverse correlation between miR-21 and Peli1 mRNA levels during liver regeneration. miR-21 overexpression in cultured cells inhibited a Peli1 3′-UTR luciferase reporter. Using NF-κB reporter assays, we determined that miR-21 overexpression inhibits NF-κB signaling. In conclusion, miR-21 expression was upregulated during early stages of liver regeneration. Targeting of Peli1 by miR-21 could potentially provide the basis for a negative feedback cycle regulating NF-κB signaling.