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Semiconductor nanomaterials like TiO2 nanoparticles (TiO2-NPs) approximately less than 100 nm in diameter have become a new generation of advanced materials due to their novel and interesting optical, dielectric, and photo-catalytic properties. With the increasing use of NPs in commerce, to date few studies have investigated the toxicological and environmental effects of NPs. Motivated by the importance of TiO2-NPs that may contribute to the cancer research field especially from the treatment prospective together with the fractal analysis technique, we have investigated the effect of TiO2-NPs on colony morphology in the dark condition using fractal dimension as a key morphological characterization parameter. The aim of this work is mainly to investigate the cytotoxic effects of TiO2-NPs in the dark on the growth of human cervical carcinoma (HeLa) cell colonies from morphological aspect. The in vitro studies were carried out together with the image processing technique and fractal analysis. It was found that, these colonies were abnormal in shape and size. Moreover, the size of the control colonies appeared to be larger than those of the treated group. The mean Df +/- SEM of the colonies in untreated cultures was 1.085±0.019, N= 25, while that of the cultures treated with TiO2-NPs was 1.287±0.045. It was found that the circularity of the control group (0.401±0.071) is higher than that of the treated group (0.103±0.042). The same tendency was found in the diameter parameters which are 1161.30±219.56 μm and 852.28±206.50 μm for the control and treated group respectively. Possible explanation of the results was discussed, though more works need to be done in terms of the for mechanism aspects. Finally, our results indicate that fractal dimension can serve as a useful feature, by itself or in conjunction with other shape features, in the classification of cancer colonies.