Epididymo-orchitis is characterized by painful swelling and inflammation of the epididymis and testes. A 4-year-old male rhesus monkey has clinical manifestations including anorexia, weakness, as well as redness and swelling of the right testis and epididymis. Bacterial culture of the urine was positive for Escherichia coli. Due to the antibiotic treatment being ineffective, the animal underwent a right orchiectomy. Postoperative histopathology confirmed necrosis and significant inflammatory cell infiltration, indicating severe acute epididymo-orchitis (EO) caused by E. coli infection in the rhesus monkey. After surgical treatment and postoperative care, the animal survived. To our knowledge, this represents the first report of acute EO caused by E. coli in a rhesus monkey. These findings not only enhance our understanding of EO in NHPs but also provide a reference for further biomedical studies of EO induced by E. coli.