Objective To investigate whether miR-100-5p regulates the proliferation and apoptosis of acute myeloid leukemia (AML) cells by targeting Tribbles pseudokinase 1 (TRIB1). Methods Peripheral blood was collected from 16 healthy children (control group) and 16 children with AML (AML group). HL-60 cells were divided into seven groups: blank, mimic negative control (mimic NC), miR-100-5p mimic, small interfering RNA negative control (si-NC), si-TRIB1, miR-100-5p mimic + TRIB1 overexpression plasmid negative control (OE-NC), and miR-100-5p mimic + TRIB1 overexpression plasmid (OE-TRIB1). The expression of miR-100-5p and TRIB1 mRNA in peripheral blood and HL-60 cells was detected by quantitative real-time PCR. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) staining and the cell counting kit-8 (CCK-8) assay (OD₄₅₀). Apoptosis was analyzed by flow cytometry. Protein levels of TRIB1, proliferating cell nuclear antigen (PCNA), p53, programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1) were determined by Western blot. The targeting relationship between miR-100-5p and TRIB1 was validated by a dual-luciferase reporter assay. Results Compared with the control group, miR-100-5p expression was decreased and TRIB1 mRNA expression was increased in the peripheral blood of the AML group (P<0.05). Compared with the blank and mimic NC groups, the miR-100-5p mimic group showed higher miR-100-5p expression, apoptosis rate, and p53 protein, and lower TRIB1 mRNA expression, EdU-positive rate, OD₄₅₀ value, and TRIB1, PCNA, PD-1, and PD-L1 proteins (P<0.05). Compared with the blank and si-NC groups, the si-TRIB1 group exhibited reduced TRIB1 mRNA expression, EdU-positive rate, OD₄₅₀ value, and TRIB1, PCNA, PD-1, and PD-L1 proteins, together with increased apoptosis rate and p53 protein (P<0.05). Compared with the miR-100-5p mimic and miR-100-5p mimic + OE-NC groups, the miR-100-5p mimic + OE-TRIB1 group had elevated TRIB1 mRNA expression, EdU-positive rate, OD₄₅₀ value, and TRIB1, PCNA, PD-1, and PD-L1 proteins, and reduced apoptosis rate and p53 protein (P<0.05). Compared with the TRIB1-WT + mimic NC group, luciferase activity was decreased in the TRIB1-WT + miR-100-5p mimic group (P<0.05). Conclusions Overexpression of miR-100-5p inhibits proliferation and induces apoptosis of HL-60 cells by downregulating TRIB1.