Background To report the clinical and microbiological features of Acanthamoeba keratitis (AK) related to contact lens use in a tertiary hospital in China. Methods In this retrospective study, the medical results of 61 cases of AK related to contact lens use from January 2000 to December 2017 were reviewed. The data included patients' demographics, lens type, history, risk factors, disease stages, corneal scraping and culture reports, and treatments. Moreover, genotypic identification of some of the isolates was carried out with a PCR assay and sequence analysis of the 18S ribosomal DNA gene. Results There were 64 eyes included in the study. A total of 32.8% of the patients wore soft contact lenses, and 67.2% of patients used overnight orthokeratology. In the cases (20 eyes) in the early stage, 65% (13 eyes) had positive results according to Giemsa-stained smears, and 0.9% sodium chloride (NaCl) wet mounts revealed trophozoites in 7 eyes (35%). Six eyes (30%) were diagnosed by confocal microscopy combined with clinical signs. In the orthokeratology patients, 87.8% (36/41) rinsed their lenses and/or cases with tap water; 55% of soft-lens wearers wore their lenses while showering. The genotype of 9 isolates was determined, and all the strains belonged to genotype T4. In the orthokeratology group, the number of patients who required therapeutic penetrating keratoplasty after 2005 was less than that before 2005 (chi-square test, chi(2) = 4.209, P = 0.04). Conclusions More than two-thirds of the cases were associated with orthokeratology. Examinations with Giemsa-stained smears, 0.9% NaCl wet mounts and confocal microscopy should be performed for patients who are highly suspected of having early-stage AK to help with early diagnosis. In the orthokeratology group, the rate of therapeutic keratoplasty after 2005 was less than that before 2005.