Introduction: The aim of this study was to evaluate the cytotoxic effects of orthodontic mini-implants on gingival fibroblasts and osteoblasts. Methods: The orthodontic mini-implants used in this study were Orthodontic Mini Implant (Leone, Florence, Italy), MTN (MTN, Istanbul, Turkey), AbsoAnchor (Dentos, Daegu, South Korea), IMTEC Ortho (3M Unitek, IMTEC, Ardmore, Okla), VectorTAS (Ormco, Glendora, Calif). The materials were incubated in Dulbecco's modified eagle's culture medium for 72 hours according to ISO 10993-5 standards (surface area-to-volume ratio of the specimen to cell-culture medium, 3 cm 2/mL). A real-time cell analyzer (xCELLigence, Roche Applied Science, Mannheim, Germany; ACEA Biosciences, San Diego, Calif) was used to evaluate cell survival. After seeding 200 μL of the cell suspensions into the wells of the E-plate 96, gingival fibroblasts were treated with bioactive components released by the metallic materials and monitored every 15 minutes for 190 hours. For the proliferation experiments, the statistical analyses used were 1-way analysis of variance and Tukey-Kramer multiple comparisons tests. Results: There was no significant differences between the human gingival fibroblast cell indexes of the control and study groups (P >0.05). When evaluated at 27 and 96 hours, only the VectorTAS mini-implants showed statistically significant decreases in the M3T3 cell index (P <0.001) compared with the control group. No significant differences were found among the control and all study groups (P >0.05). Furthermore, the Leone and MTN mini-implants showed statistically significant decreases (P <0.001) at 190 hours. Also, the VectorTAS mini-implants demonstrated a significant decline (P <0.05) at the same time in the M3T3 cell index. Conclusions: These findings provide fundamental knowledge and new insights for future design and development of new biocompatible titanium alloys for orthodontic mini-implants and temporary anchorage devices. © 2012 by the American Association of Orthodontists.