Prohibitin (PHB) was originally isolated and characterized as an anti-proliferative gene in rat liver. The evolutionarily conserved PHB gene encodes two human protein isoforms with molecular weights of ~33 kDa, PHB1 and PHB2. PHB1 and PHB2 belong to the prohibitin domain family, and both are widely distributed in different cellular compartments such as the mitochondria, nucleus, and cell membrane. Most studies have confirmed differential expression of PHB1 and PHB2 in cancers compared to corresponding normal tissues. Furthermore, studies verified that PHB1 and PHB2 are involved in the biological processes of tumorigenesis, including cancer cell proliferation, apoptosis, and metastasis. Two small molecule inhibitors, Rocaglamide (RocA) and fluorizoline, derived from medicinal plants, were demonstrated to interact directly with PHB1 and thus inhibit the interaction of PHB with Raf-1, impeding Raf-1/ERK signaling cascades and significantly suppressing cancer cell metastasis. In addition, a short peptide ERAP and a natural product xanthohumol were shown to target PHB2 directly and prohibit cancer progression in estrogen-dependent cancers. As more efficient biomarkers and targets are urgently needed for cancer diagnosis and treatment, here we summarize the functional role of prohibitin domain family proteins, focusing on PHB1 and PHB2 in tumorigenesis and cancer development, with the expectation that targeting the prohibitin domain family will offer more clues for cancer therapy.