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This in vitro study aimed to investigate the effect of plasma ion implantation nitriding on titanium's surface properties and initial cell response. Grade 4 titanium discs (12.7 × 2 mm) were blasted with aluminum oxide particles to create moderately rough surfaces. The experimental discs (TiN) were nitrided using the plasma ion implantation technique in a vacuum chamber. Surface characterization was performed using laser confocal microscopy, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Surface wettability was assessed by measuring the contact angle of a sessile drop using a goniometer. Human osteoblast cells were seeded on the discs to evaluate cell attachment and proliferation at 1, 3, 5, and 7 days of culture using a tetrazolium compound assay. Alkaline phosphatase (ALP) activity was measured at day 7 to assess cell differentiation. Cell morphology was examined by scanning electron microscopy (SEM) and confocal laser scanning microscopy. The TiN group exhibited similar micro-roughness to the control group; however, it displayed a higher density of nanostructures, increased nitrogen content, and slightly improved wettability. Cell proliferation and ALP activity were similar between the groups after seven days of culture. In conclusion, plasma ion implantation nitriding enhances surface nanofeatures and wettability without compromising the biocompatibility of titanium, making it a promising surface modification technique for dental and orthopedic implants.