Mesenchymal stem cells (MSCs) represent a promising tool for tissue engineering and regenerative medicine. However, their procurement often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their sampling and further processing are non-invasive for donors. The goal of this study was to analyze their morphology and selected biologic properties including the kinetics of proliferation, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm2, they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed two distinct cell populations. The first type display a rice-grain shape and the second one was characterized by a polyhedral morphology. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. On the other hand, they were negative for markers of hematopoietic and endothelial cells. We have successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. Based on the mentioned above it can be emphasized that UDSCs might have immense potential in tissue engineering and in the regeneration of damaged tissues.