Diffuse large B cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma in China. 1,25-Dihydroxyvitamin D-3 [1,25(OH)(2)D-3] has been shown to possess significant antitumor potential and is degraded by 25-hydroxyvitamin D-24-hydroxylase (CYP24A1). In the present study, the role of CYP24A1 and autophagy, and their underlying mechanisms in the anticancer effects of 1,25(OH)(2)D-3 in DLBCL cells, were investigated. It was found that the levels of CYP24A1 in DLBCL lymph node tissues were higher than in hyperplasia lymphadenitis tissue. Moreover, the expression of CYP24A1 was positively associated with the Ann Arbor stage and the International Prognostic Index in patients with DLBCL, and negatively associated with the clinical response to treatment. Patients >60 years of age were found to have a higher level of CYP24A1. 1,25(OH)(2)D-3 inhibited the proliferation of the Pfeiffer DLBCL cell line and increased the G1 phase population of Pfeiffer cells. Rapamycin (RAPA) in combination with 1,25(OH)(2)D-3 increased the G1 phase distribution of Pfeiffer cells. Furthermore, RAPA blocked the increase of CYP24A1 and vitamin D receptor (VDR) expression induced by 1,25(OH)(2)D-3. 1,25(OH)(2)D-3 induced the formation of autophagosomes, increased the expression of autophagy related protein light chain (LC)3II/LC3I and reduced the expression of the ubiquitin binding protein P62. In addition, 1,25(OH)(2)D-3 decreased the phosphorylation of AKT and mammalian target of RAPA (mTOR), and downstream targets eukaryotic translation imitation factor 4E-binding protein 1 and ribosomal protein S6 kinase beta-1 in Pfeiffer cells. The results from the present study suggested that CYP24A1 may be a novel prognostic indicator for DLBCL. 1,25(OH)(2)D-3 inhibited proliferation and induced autophagy of Pfeiffer cells. In addition, 1,25(OH)(2)D-3 increased the G1 phase population of Pfeiffer cells. These effects may be mediated by inhibition of the AKT/mTOR/PI3K signaling pathway. RAPA increased the cell cycle arrest induced by 1,25(OH)(2)D-3 by blocking the upregulated expression of CYP24A1 and VDR.