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NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF-7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)-9 were assessed by reverse transcription-quantitative PCR and western blotting, and the activity of MMP-9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and invasion of MCF-7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA-induced MMP-9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA-induced MMP-9 expression and invasion in MCF-7 cells. NOX1- and NOX5-specific small interfering RNAs attenuated TPA-induced MMP-9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA-induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA-induced intracellular ROS levels, MMP-9 expression and NOX activity in MCF-7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA-induced MMP-9 expression and invasion of MCF-7 breast cancer cells. Furthermore, the present study indicated that TPA-induced MMP-9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti-invasive effects in breast cancer. [ABSTRACT FROM AUTHOR]