The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.