AIM: To evaluate the efficacy and safety of intrastromal transplantation of adipose-derived stem cells (ASCs) in keratoconus patients. METHODS: This study was conducted on 8 eyes of 8 patients with moderate to severe keratoconus. In the patients, ophthalmic assessments including visual acuity, refraction, slit lamp examination, fundoscopy, corneal topography, and confocal microscopy were performed. Autologous stem cells were used. The isolated stem cells were injected into the corneal stroma by using femtosecond laser. Surgical procedure was similar to intracorneal ring implantation. All patients were re-assessed 1, 3, and 6mo after surgery. RESULTS: The baseline mean visual acuity was 0.48±0.18 and improved to 0.66±0.17 after surgery and final acuity increased by 1.85±0.80 lines (P=0.001). The mean spherical refraction of patients improved 0.34±0.35 D (P=0.039), and the mean cylindrical refraction of patients improved 0.84±0.23 D (P=0.016). The mean flat keratometry decreased 0.78±0.71 D (P=0.017), and the mean steep keratometry decreased 0.59±0.68 D (P=0.023). The mean central corneal thickness of patients improved of 6.29±4.47 μm (P=0.03). The mean keratocyte density at the anterior and middle stroma of cornea increased (P<0.05) but remained stable at the posterior stroma after 6mo. All patients had no complications and their corneas remained transparent. CONCLUSION: Intrastromal transplantation of ASCs has positive effects on vision and refractive parameters in most patients with keratoconus. After six months, visual acuity improved moderately, corneal parameters reduced slightly, and stromal keratocytes density increased. This modality is safe, and patients do not have any complications.