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The aim of the present study was to evaluate<jats:italic>in vitro<\/jats:italic>and<jats:italic>in vivo<\/jats:italic>bioactivities of a multilayered DNA\/protamine (D\/P) coating on Ti implant by simulated body fluid (SBF) immersion experiments and implantation experiments into extracted sockets of rat molars. Two types of DNA, 300 base pair (bp) and 7000 bp fragments, were used. Protamine was initially immobilized onto Ti implants using a tresyl chloride\u2010activated method and DNA and protamine were then alternatively deposited after the immobilization of protamine by a layer\u2010by\u2010layer technique. A multilayered D\/P\u2010coating was confirmed by X\u2010ray photoelectron spectroscopy and Fourier\u2010transform infrared spectroscopy measurements. The deposition of apatite progressed more on the surfaces of multilayered D\/P\u2010coated Ti implants than on those of nontreated Ti implants in SBF immersion experiments. Animal implantation experiments showed that multilayered D\/P\u2010coated Ti implants provided a significantly higher bone\u2010to\u2010implant (BIC) contact ratio 3 weeks after implantation. No significant difference was observed in the BIC ratio 9 weeks after implantation. The results of the present study demonstrated that a multilayered D\/P\u2010coating promoted new bone formation at the early stages of the bone healing process. \u00a9 2016 Wiley Periodicals, Inc. 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