Neuroblastoma is the primary cause of cancer-related death for children 1 to 5 years of age. New therapeutic strategies and medicines are urgently needed. This study aimed to investigate the effects of triptolide (TPL), the major active component purified from Tripterygium wilfordii Hook F, on neuroblastoma SH-SY5Y cell proliferation, migration, and apoptosis, as well as underlying potential mechanisms. We found that TPL inhibited SH-SY5Y cell viability, proliferation, and migration, but induced cell apoptosis. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 after TPL treatment in SH-SY5Y cells was decreased. The expression of microRNA-181a (miR-181a) was upregulated after TPL treatment. Moreover, suppression of miR-181a reversed the effects of TPL on SH-SY5Y cell proliferation, apoptosis, and migration. Overexpression of miR-181a enhanced the TPL-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathways. In conclusion, our research verified that TPL inhibited the proliferation and migration of human neuroblastoma SH-SY5Y cells by upregulating the expression of miR-181a.