The therapeutic potential of mesenchymal stem cells (MSCs) and their conditioned medium (MSC-CM) has been extensively studied. MSCs can repair tissue, reduce local inflammation, and modulate the immune response. Persistent renal tubular interstitial inflammation results in fibrosis and leads to chronic kidney disease (CKD). Unilateral ureteral obstruction (UUO) is a very well-accepted renal fibrosis model. In this study, we evaluated factors influenced by the administration of MSCs or MSC-CM in the UUO model. MSCs extracted from rat bone marrow were cultivated in vitro and characterized by flow cytometry and cellular differentiation. Eight groups of female rats were used in experiments (n = 7, each), including Sham, UUO, UUO + MSC (obstruction + MSC), and UUO + CM (obstruction + MSC-CM) for 7 days of obstruction and Sham, UUO, UUO + MSC, and UUO + CM for 14 days of obstruction. The MSCs or MSC-CM was administered via the abdominal vena cava after total ligation of the left ureter. After 7 or 14 days, rats were euthanized, and serum and obstructed kidney samples were collected. MSCs or MSC-CM decreased the expression of molecules, such as Col1a1, α-SMA, and TNF-α. We also observed reductions in the levels of caspase 3, α-SMA, and PCNA in treated animals by immunohistochemistry. Our results suggest that the intravenous administration of MSCs or MSC-CM improves fibrosis progression and factors involved in apoptosis, inflammation, cell proliferation, and epithelial‐mesenchymal transition in Wistar rats subjected to UUO, indicating a potential tool for preventing CKD.