Piperlongumine (PL), a natural product derived from long pepper (Piper longum L.), is known to exhibit anticancer effects. However, the effect of PL on cell cycle-regulatory proteins in estrogen receptor (ER)-positive breast cancer cells is unclear. Therefore, we investigated whether PL can modulate the growth of ER-positive breast cancer cell line, MCF-7. We found that PL decreased MCF-7 cell proliferation and migration. Flow cytometric analysis demonstrated that PL induced G2/M phase cell cycle arrest. Moreover, PL significantly modulated the mRNA levels of cyclins B1 and D1, cyclin-dependent kinases 1, 4, and 6, and proliferating cell nuclear antigen. PL induced intracellular reactive oxygen species (hydrogen peroxide) accumulation and glutathione depletion. PL-mediated inhibition of IKKβ expression decreased nuclear translocation of NF-κB p65. Furthermore, PL significantly increased p21 mRNA levels. In conclusion, our data suggest that PL exerts anticancer effects in ER-positive breast cancer cells by inhibiting cell proliferation and migration via ROS accumulation and IKKβ suppression.