MiR-221 is frequently upregulated in papillary thyroid cancer (PTC) tissues and cell lines, and this study was designed to validate the association of miR-221 with PTC proliferation, apoptosis, and migration. We observed that miR-221 suppressed TIMP3 expression by binding to 3′ untranslated region of TIMP3 mRNA, and TIMP3 expression was increased with the presence of miR-221 inhibitors; TIMP3 siRNA could reverse the effects of miR-221 inhibitors on PTC cells. The results indicated that miR-221 exacerbated PTC by downregulating the expression of TIMP3. The effects of miR-221 and TIMP3 in vivo were also confirmed by human PTC-bearing mice models which suggest consistent results with those in vitro studies. In summary, miR-221 could aggravate cell proliferation and invasion of PTC by targeting TIMP3.