Pseudomonas aeruginosa, the opportunistic pathogen, occupies one of the leading places in the structure of pathogens causing nosocomial infections, which is due to high adaptive potential and the ability to quickly develop antimicrobial resistance. The study aimed to assess the influence of the P. aeruginosa adaptation to colistin on bacterial fitness. A total of nine isolates obtained during the experimental evolution of the P. aeruginosa strain (laboratory number 1202) under conditions of increasing colistin concentrations, the growth kinetics of which was compared to that of wild type strain, were included in the study; the whole genome sequencing of all isolates was performed, and the minimum inhibitory concentration of colistin was determined. Growth rate was estimated using the Varioskan LUX multimodal reader (Thermo Scientific, USA) throughout 18 h at 37 °С; optical density (OD) at λ = 600 nm was measured every 15 min. The maximum growth rate (GR_max, i.e. the maximum change in OD within 1h) and the time to reach 50% of the maximum OD reported when growing the wild type Ра_1202_0 strain (T_OD_50%) were considered. Isolates of the clone carrying mutations of the genes phoQ, lptA, and prs showed low fitness values compared to wild type strains. For example, GR_max of the isolate Ра_1202_43 was 0.029 OD/h vs. 0.182 OD/h reported for the original isolate Ра_1202_0, and it reached OD_50% 4.6 h later. The growth characteristics of the clone carrying mutations of lpxL and lptB, as well as the clone carrying mutant pmrB were generally comparable with the characteristics of the wild type strain. Thus, the genome modifications observed during the P. aeruginosa adaptation to colistin have an ambiguous effect on bacterial fitness.