OBJECTIVE: To report four cases of microsporidial keratoconjunctivitis (MKC) from The Affiliated Eye Hospital of Nanjing Medical University (from May 2023 to October 2024) and to aid ophthalmologists in diagnosing and treating MKC, as MKC has been increasingly reported in Asian healthy individuals but not much in Mainland China. METHODS: Four patients with MKC were studied. Demographic information, symptoms, and clinical data were collected. Diagnosis involved ophthalmic examinations, corneal scraping microscopy (including Giemsa staining, modified Ziehl-Neelsen staining, Calcofour white staining), bacterial and fungal cultures, and metagenomic next-generation sequencing (mNGS). Treatment included various topical medications like polyhexamethylene biguanide (PHMB), fluconazole, tacrolimus, sodium hyaluronate, and systemic medication such as albendazole. RESULTS: Three cases were caused by Encephalitozoon hellem proved by mNGS. Patients had symptoms like eye redness, swelling, pain, foreign body sensation, and vision loss. Risk factors included improper contact lens - wearing habits, contact with birds, or exposure to potentially contaminated environments. All patients showed improvements after treatment, with 3 cases cured and 1 case improved. CONCLUSION: MKC is a unilateral, acute, non-purulent ocular surface infectious disease. Clinicians should be more aware of it. Diagnosis depends on recognizing clinical signs, exploring risk factors, and laboratory tests. There is no consensus on treatment, but combined topical and systemic anti-protozoal drugs showed good results. Further large-scale validation is needed. Relevant departments should strengthen water source management, and patients should pay attention to personal hygiene.