PURPOSE: This study aimed to explore the effect of GANT61 on regulating cell proliferation, cell apoptosis and cell cycle, and to investigate whether GANT61 would function in multiple myeloma (MM) via inhibiting Notch pathway. : RPMI-8226 and U266 cells were treated by GANT61 (0, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 μmol/L) for 18, 24 and 36 hours (h), and cell proliferation was detected by Cell Counting Kit 8. Then these cells were treated by GANT61 at 0, 2.5, 5.0, 10.0 μmol/L for 24 h or treated by 10.0 μmol/L GANT61 for 0, 18, 24 and 36 h, and cell apoptosis rate, apoptosis markers and cell cycle were detected by AV/PI, Western blot, and PI staining. Notch1, Jagged1, Jagged2 and Hes1 expressions were detected by qPCR and Western blot. Further rescue experiments were conducted by upregulating Notch1. : In RPMI-8226 and U266 cells, GANT61 inhibited cell proliferation, increased cell apoptosis rate and cell percentage of G1/G0 phase while decreased cell percentage of S phase in a dose- and time-dependent manner. Besides, GANT61 inhibited Notch1, Jagged1, Jagged2 and Hes1 expressions in a dose- and time-dependent manner as well. In rescue experiments, Notch1 upregulation attenuated the inhibition of cell proliferation, promotion of cell apoptosis, induction of G1/G0 cycle retardation and repression of Notch signaling pathway induced by GANT61 treatment in RPMI-8226 and U266 cells. : GANT61 suppresses cell proliferation, promotes cell apoptosis and induces G1/G0 cycle retardation with a dose- and time-dependent manner through inhibiting Notch pathway in MM. ABBREVIATIONS: MM: Multiple myeloma; Hh: Hedgehog; EMT: epithelial mesenchymal transition; AML: acute myeloid leukemia; GANT61: GLI antagonist; DMSO: dimethyl sulfoxide; CCK-8: Cell Counting Kit 8; C-Caspase 3: Cleaved Caspase 3; Bcl-2: B-cell lymphoma-2; RT-qPCR: real-time quantitative polymerase chain reaction; OD: optical density; PTCH1: Patched1.