Background: Breast cancer (BC) is the most common type of cancer in women. It is urgent to identify new therapeutic targets and their mechanisms. Platelet activating factor acetylhydrolase 1B3 (PAFAH1B3) is a multimeric enzyme which is a vital metabolic enzyme that mediates lipid metabolism and affects several tumors. This study was performed to elucidate the function of PAFAH1B3 in BC progression and investigate its underlying mechanisms. Methods: Gene Expression Profiling Interactive Analysis (GEPIA) database and immunoblot showed the expression of PAFAH1B3 in breast cancer tissues. cell counting kit-8 (CCK-8), colony formation, and transwell assays showed the effects on breast cancer cell growth and migration. Flow cytometry (FCM) and immunoblot assays exhibited the effects on the apoptosis of breast cancer cells. Mechanically, immunoblot was further conducted to confirm the mechanism. Results: Our findings reveal that PAFAH1B3 is highly expressed in BC and that the depletion of PAFAH1B3 inhibits BC cell growth and migration while promoting apoptosis. Mechanistically, PAFAH1B3 depletion disrupts the Phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (AKT) pathway, thereby suppressing BC progression. Conclusions: We found that PAFAH1B3 enhances BC cell growth as well as motility via the PI3K/AKT axis and could be a target for BC.