Objective To explore the regulation and mechanism of prolyl 4-hydroxylase β-polypeptide(P4HB) on the biological function of acute myeloid leukemia(AML) cells. Methods The mRNA and protein expression levels of P4HB in human peripheral blood mononuclear cells (hPBMC) and AML cell lines THP-1, HL-60, U937, MOLM-13, SKNO-1 were detected by RT-qPCR and Western blot. MOLM-13 cells were divided into control group, si-NC group and si-P4HB group, and si-NC or si-PHB cells were transfected into si-NC or si-PHB group with Lipofectamine TM 2000 transfection reagent, respectively. The cell proliferation activity was detected by MTT assay, number of cell clones formation was detected by plate clone formation assay. The level of cell apoptosis was detected by TUNEL, the distribution of cell cycle was detected by flow cytometry, and the level of cell invasion and migration were evaluated via Transwell. The P4HB gene expression level was detected by RT-qPCR. The protein expression levels of CDK1, PCNA, p21, Bax, caspase-3, Bcl-2, p-ERK, ERK, p-p38, p38, p-JNK and JNK were detected by Western blot. Results Compared with hPBMC cells, mRNA and protein expression levels of P4HB in AML cell lines THP-1, HL-60, U937, MOLM-13 and SKNO-1 were increased(P<0.05). Compared with the control group or the si-NC group, the cell proliferation activity, number of clones formation, migration and invasion cells in the si-P4HB group were decreased(P<0.05), and the TUNEL-positive cell rate and the proportion of G2/M phase cells were increased(P<0.05). The protein expression levels of Bax, caspase-3 and p21 were increased(P<0.05), while the protein expression levels of Bcl-2, CDK1 and PCNA were decreased(P<0.05). The p-ERK/ERK ratio decreased, the p-p38/p38 ratio increased(P<0.05), and the p-JNK/JNK ratio had no statistical significance(P>0.05). Conclusion P4HB is highly expressed in AML cells, and interference with the expression of P4HB can inhibit the proliferation, migration and invasion of AML cells, promote apoptosis and G2/M phase arrest of AML cells. The regulatory effect of P4HB on AML cells may be related to the inhibition of ERK subpathway and the activation of p38 subpathway in MAPK signaling pathway