Purpose: To report 28 cases of infectious keratitis related to orthokeratology lens overnight wear in China. Methods: From March 2000 to August 2001, 28 cases of infectious keratitis related to overnight orthokeratology lens wear were diagnosed in Beijing Institute of Ophthalmology. These were retrospectively reviewed with regard to the pathogens isolated, duration of wear, the time since onset of symptoms, and age. Cultures of corneal scrapes for bacteria, fungus and Acanthamoeba were performed in all of the 28 cases. Results: All cases were students, including 10 males and 18 females, average age was 16 years (range 10-21 years). The duration of orthokeratology overnight wearing was from 2 weeks to 2 years. Uncorrected visual acuity (UCVA) on initial examination in our institute was from 20/200 to light perception. Of 28 isolates, 24 were culture positive (including 11 bacteria, 11 Acanthamoeba and two fungi), and four were culture negative. In two of the four culture negative cases, Acanthamoeba cysts were detected in the corneal stroma with the confocal microscope. Acanthamoeba and Pseudomonas aeruginosa accounted for 75% (21 of 28) of the cases of infectious keratitis. Conclusion: Infectious keratitis is a severe complication associated with overnight orthokeratology lens wear. Ophthalmologists should pay more attention to this complication in practice.