Aim Management of osteoarthritis (OA) is basically symptomatic. Recently, stem cells (SC) have been used in the search for an optimum treatment. We decided to conduct a controlled clinical trial to determine if a single intra‐articular injection of in vivo stimulated bone marrow SC could lead to an improvement in pain management and quality of life in patients with knee OA. Method This was a prospective, open‐label, phase I/II clinical trial to assess the safety and efficacy of a single intra‐articular injection of autologous stimulated bone marrow stem cells (BM‐SC) in patients with knee OA. Individuals of both genders older than 30 years with confirmed diagnosis of OA who signed informed consent were included in two groups: SC group received in vivo BM stimulation with subcutaneous administration of granulocyte colony stimulating factor (G‐CSF). SC were obtained by BM aspiration and administered in a single intra‐articular injection. The control group received exclusively oral acetaminophen. Visual analogue scale and Western Ontario and McMaster Universities Osteoarthritis Index scores were performed at 1 week, 1 month and 6 months in both groups. This trial was registered in ClinialTrials.gov NCT01485198. Results A total of 61 patients were included. Socio‐demographic characteristics, OA grades and initial scores were similar in both groups. The BM‐SC group showed significant improvement in knee pain and quality of life during the 6‐month follow‐up. Conclusion The study demonstrates feasibility and supports efficacy of a completely ambulatory procedure in treatment of knee OA.