RORA plays an important role in regulating circadian rhythms, inflammation, metabolism and cellular development. Herein, we explore the roles of Rora in B cell proliferation and differentiation, as well as in Ph+ B-ALL. By using Roraloxp/loxp Mx-1-Cre mice, Rora was deleted in hematopoietic cells post Pipc induction. Rora deficiency mice were associated with an obvious accumulation of B cells in the peripheral blood, bone marrow, and spleen. On the other hand, activation of Rora with Cholesterol sulfate (CS) was associated with decreased B cell numbers. RNA-seq analysis revealed that the transcription level of Lmo1 was decreased in Rora deficient B cells. Moreover, the expression of RORA was shown to be decreased in Ph+ B-ALL cells compared to peripheral blood derived B cells from healthy donors. The overexpression of Rora in BaF3 cells with BCR/ABL1 was also associated with impeded the cell growth and an increased apoptotic rate compared to cells transduced with BCR/ABL1 alone. The co-expression of BCR/ABL1 and Rora induced B-ALL mouse model was associated with the significant inhibition of BCR/ABL1-transformed cell growth and prolonged the survival of the diseased mice. These results suggest a novel role for Rora in B cell development and Ph+ leukemogenesis.© The author(s).