Fibroblasts are the effector cells of collagen secretion in renal interstitial fibrosis (RIF), and their proliferation and activation are essential for the development of RIF. Hypoxic ischemia in local tissues has been identified in chronic kidney diseases (CKDs), with adenosine (ADO) as a key signaling molecule. The current study investigated the association between ADO and the biological behavior of renal fibroblasts by establishing an in vitro hypoxia cell model. This aimed to provide experimental evidence for the prevention and treatment of RIF. NIH3T3 fibroblasts were exposed to hypoxia, and the subtypes of the ADO receptor (AR) on the cell surface were identified by a TaqMan probe‑based assay. Cells were divided into the following four groups: i) Control; ii) 5'‑N‑ethylcarboxamidoadenosine (NECA); iii) PT, NECA + 8‑phenyltheophylline (PT); and iv) MRS, NECA + N‑(4‑cyanophenyl)‑2‑[4‑(2,3,6,7‑tetrahydro‑2,6‑dioxo‑1,3‑dipropyl‑1H‑purin‑8‑yl)phenoxy]‑acetamide (MRS1754). The mRNA levels of transforming growth factor‑β1 (TGF‑β1), procollagen α1 (I) and α‑smooth muscle actin (α‑SMA) were measured following 24, 48, and 72 h of hypoxia. Cell proliferation was evaluated by a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay at 0, 12, 24, 48 and 72 h. The results demonstrated that A2BR was the predominant AR subtype present in hypoxia‑stimulated fibroblasts. NECA significantly induced fibroblast proliferation and upregulated the expression of TGF‑β1, procollagen α1 (I) and α‑SMA mRNA, while 8‑PT and MRS1754 inhibited fibroblast proliferation and downregulated the expression of TGF‑β1, procollagen α1 (I) and α‑SMA mRNA. The blockage of A2BR in hypoxia significantly inhibited the proliferation and activation of fibroblasts, and reduced the production of profibrotic cytokines, thus preventing the generation and development of fibrosis.