Multiple myeloma (MM) is a hematological malignancy with a high prevalence and is characterized by the clonal expansion of malignant plasma cells. As a new tumor suppressor, defective in sister chromatid joining (DIS3) was reported to be a gene closely related to MM. This study elucidated the biological functions and underlying mechanisms of DIS3 in MM. DIS3 mRNA and protein levels were detected using RT-qPCR and western blotting, respectively. Methyl thiazolyl tetrazolium assays, flow cytometry analyses, Transwell assays, and wound healing assays were performed to detect the proliferation, apoptosis, invasion, and migration of MM cells. The binding relationship between miR-125a/b-5p and DIS3 was verified using luciferase reporter assays and RNA pulldown assays. Xenograft tumor models were established in nude mice to investigate the effects of miR-125a/b-5p and DIS3 on tumor growth in vivo. DIS3 levels were downregulated in MM cells, and DIS3 upregulation inhibited the malignant behaviors of MM cells. Mechanistically, miR-125a/b-5p directly targeted the 3' untranslated region of DIS3. The expression of miR-125a/b-5p was upregulated in MM cells, miR-125a/b-5p knockdown inhibited the malignant behaviors of MM cells, and the inhibitory effect was reversed by DIS3 downregulation. The results of in vivo experiments indicated that miR-125a/b-5p promoted tumor growth by downregulating DIS3. Overall, miR-125a/b-5p promotes MM cellular processes and xenograft tumor growth by targeting DIS3.