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Mycoplasma hominis is the facultative-pathogenic cell wall-less bacterium that is found as a commensal in the urogenital tract. The metabolic capabilities of M. hominis are reduced and seem to be adapted to their natural niche. M. hominis is nonglycolytic species and produces energy through arginine degradation. The recent data suggested that M. hominis can utilize ribose-phosphate and deoxyribose-phosphate, particularly thymidine, formed by nucleoside catabolism as energy sources. The aim of our work was to find out how the utilization of different energy sources can affect the physiology of M. hominis. We analyzed growth rate, antibiotic sensitivity, and biofilm formation of M. hominis grown on thymidine and arginine- supplemented medium. We showed that growth on thymidine led to a slow-growth phenotype of bacteria. These cells demonstrated reduced susceptibility to some antibiotics, and increased extent of biofilm formation. Comparative proteomic analysis of M. hominis grown on thymidine and arginine allowed us to outline the molecular basis of slow-growth phenotype and suggest possible regulators. The energy source strongly affected the synthesis of enzymes related to RNA modifications and ribosome assembly as well as translocation machinery and abundance of membrane-associated proteins. This study can help to find out mechanisms underlying the interaction between growth rate and fitness of M. hominis.