Matrine is a natural product extracted from the root of Sophora flavescens that has been shown to be a promising alternative drug in different types of cancer. Here, we aimed to investigate the antitumor effects of matrine on human papillary thyroid cancer (PTC) and explore the underlying molecular mechanisms. Our data demonstrated the following findings. (a) The expression of miR-182-5p was significantly upregulated in PTC tissues and cell lines. (b) Matrine inhibited the expression of miR-182-5p and induced the apoptosis of TCP-1 and BCPAP cells in a dose-dependent manner. (c) Matrine increased caspase3 expression levels and reduced Bcl-2 expression levels in both TCP-1 and BCPAP cells. (d) Matrine appreciably inhibited PTC tumor growth in vivo. (e) After miR-182-5p overexpression, matrine-induced apoptosis and caspase3 activation were inhibited, and the matrine-induced decrease in Bcl-2 expression was abolished. (f) Overexpression of miR-182-5p counteracted the inhibitory effects of matrine on PTC tumor growth. In conclusion, these results demonstrate that matrine exerts antitumor effects possibly by inducing the apoptosis of TCP-1 and BCPAP cells, decreasing the level of Bcl-2, activating caspase3 and suppressing PTC tumor growth by downregulating the expression of miR-182-5p. These findings explain the anticancer mechanisms of matrine in PTC and identify miR-182-5p as an effective target of matrine in PTC treatment.