In this study, plasma electrolytic oxidation of Ti-6Al-4V alloy in electrolytes containing bone formation ions was examined using various instruments. Here, a Ti-6Al-4V ELI disk is used for PEO treatment. A pulsed DC power of 280 V was applied to all specimens for 3 min. The electrolytes used for PEO were prepared by mixing of calcium, prosperous, silicon, strontium, zinc, magnesium, and manganese ions. Additionally, MC3T3-E1 mouse osteoblasts were cultured on the specimens for the cell proliferation assay. The morphology of the attached cells was observed using field-emission scanning electron microscopy (FE-SEM). The surface characterization of the PEO-treated surface was performed by FE-SEM and energy dispersive X-ray spectroscopy. The surface area of the pores was measured using an image analyzer. Furthermore, the phase of the specimen was analyzed with a thin film X-ray diffractometer (XRD).The PEO films, except the specimen containing manganese element, exhibit a uniform porous surface. The porous structure displays a very rough surface, and Mn easily precipitates around pores in the process of original and new pore formation. A high amount of Mn was detected in the precipitate inside the pores, whereas high amounts of calcium, phosphorus, strontium, titanium, and oxygen were detected around the pores. The XRD peak of HA on PEO-treated surface in the electrolyte containing the ions of five elements is lower, broader and shifted toward the left side. The pores were covered with MC3T3-E1 cells and extended well. Cells exhibit a spinning form of filopodia in lamellipodia and grow in association with the cells on PEO-treated surface in an electrolyte containing five ions. The cell differentiation and bone formation ability of ions of five elements doped surface were significantly increased compared to bulk surface.