The role of prolyl 3-hydroxylase family member 4 (P3H4, or SC65) in breast cancer and the molecular mechanisms in which this protein was involved were investigated. For this purpose, microchips with cancerous and paracancerous tissues collected from 56 patients with breast cancer were constructed. The P3H4 protein expression was evaluated by immunohistochemistry. Cell lines with decreased and increased P3H4 expression were selected and divided into five groups: P3H4 overexpression and corresponding negative control, P3H4 knockout groups #1 and #2 and corresponding negative control. CCK8 assay, colony formation test, immunoblotting, scratch test, transwell test and flow cytometry were used to determine the relevant cell functions. P3H4 expression was higher in breast cancer cells than in paracancerous tissue. Compared with corresponding negative control, proliferative activity of the cells was inhibited at 24, 48 and 72 h, migration activity and invasion ability of the cells were reduced, autophagy and apoptosis in the cells were increased in P3H4 knockout groups #1 and #2. P3H4 knockout promoted apoptosis of breast cancer cells and inhibited their proliferation, migration, and invasion by activating the BCL-2/BAX/Caspase-3 and AMPK/mTOR pathways. P3H4 knockout promoted apparently autophagy by activating the AMPK/mTOR/ULK1 pathway. However, P3H4 overexpression could promote the proliferation, migration and invasion of breast cancer cells, and inhibited apoptosis and autophagy of mammary gland adenocarcinoma cells MDA-MB-231.