Objective: To investigate the clinicopathological features, immunological phenotype, treatment and prognosis of relapsed B-cell malignancies with loss of CD20 immunoreactivity after rituximab therapy. Methods: A retrospective analysis of one case of follicular small cleaved cell lymphoma which transformed to CD20-negative DLBCL after rituximab treatment was conducted. We reviewed and analyzed the clinicopathological features, immunological phenotype, treatment and prognosis of the patients. Results: We describe an 87-year-old male who initially presented with enlargement of left parotid lymph node in 1987. He was diagnosed with follicular small cleaved cell lymphoma through histological evaluation of left parotid lymph node biopsy in 1988. He was treated with 50 courses of cyclophosphamide, vincristine, prednisone (COP or OP), and local irradiation therapy. In 1998, he developed a high white blood cell count and an increased percentage of lymphocytes. His bone marrow aspiration simultaneously showed chronic lymphocytic leukemia. He received some cycles of rituximab treatment due to a sharp increase in white blood cells from March 2012 to March 2014. He relapsed with a sore throat in March 2014, and the biopsy of right side of epiglottis revealed CD20-negative DLBCL. Then he received 2 cycles of rituximab combined with low-dose CHOP (mini-CHOP) before the mass in the right side of the epiglottis disappeared. While he relapsed again with the enlargement of right neck lymph node in May 2015, and the lymph node biopsy indicated CD20-positive DLBCL. Conclusion: It is not rare of the loss of CD20 antigen expression in malignant B-cell lymphoma after rituximab treatment. We recommend relapsed or non-sensitive patients after therapy with rituximab to rebiopsy for pathological diagnosis, immunohistochemistry, and even molecular genetic testing if necessary. These can reduce misdiagnosis, and play an important role in adjusting therapy of relapsed patients.