Hypoxia contributes to the phenotypic switch of vascular smooth muscle cells (VSMCs). Various microRNAs (miRNAs) participate in this process as post‑transcriptional regulators, however the mechanism remains unclear. In the present study, mouse VSMCs (mVSMCs) harvested from aortas were cultured in normoxic and hypoxic conditions, and the mRNA levels of miR-26b-5p, desmin, H‑caldesmon and smoothelin were quantified using reverse transcription‑quantitative polymerase chain reaction. Following treatment with a miR‑26b‑5p antagonist (agomir) or non‑targeting control (scramble), the cell areas of normoxic and hypoxic mVSMCs were analyzed by immunofluorescence staining. In addition, the protein expression levels of collagen Iα, Smad2/phosphorylated (p)‑Smad2, Smad3/p‑Smad3 and Smad4 were determined by western blotting. Potential miRNA26b‑5p binding sequences in the 3'‑untranslated region (UTR) of Smad4 were investigated, and the distribution of Smad4 in mVSMCs was visualized using immunofluorescence methods. Hypoxic mVSMCs exhibited a significant downregulation miR‑26b‑5p, upregulation of hypoxia inducible factor‑1α mRNA and suppression of desmin, H‑caldesmon and smoothelin mRNA levels. Additionally, miR‑26b‑5p agomir reduced the cell area and decreased collagen Iα expression levels in hypoxic mVSMCs compared with normoxic mVSMCs transfected with agomir, and the area was comparable with those of normoxic mVSMCs transfected with agomir or scramble. Furthermore, miR‑26b‑5p suppressed Smad4 expression in hypoxic mVSMCs, but did not change the expression levels of Smad2 and Smad3, p‑Smad2 and p‑Smad3, however p‑Smad2 and p‑Smad3 levels were upregulated in response to hypoxic stimuli. Additionally, the miR‑26b‑5p agomir caused weak immunoreactivity with Smad4 in hypoxic mVSMCs. The binding motif of miR‑26b‑5p in the Smad4 3'‑UTR was identified as UACUUGA at position 978-984. These findings suggest that miR‑26b‑5p regulates hypoxia‑induced phenotypic switching of VSMCs via the transforming growth factor β/Smad4 signaling pathway.