MLAA-34 is a novel acute monocytic leukemia (M5)-associated antigen (MLAA) that plays a role in the apoptosis of U937 cells. However, the expression and molecular mechanism of MLAA-34 in U937 cells remain largely unclear. Here, we utilized three strategies to gain insight into the expression and molecular functions of MLAA-34 and to identify its interacting proteins and pathways involved in the fine-tuning of the MLAA-34 response. Western blot analysis was performed to assess the expression of MLAA-34 in 41 cell lines and five mixed cell types, which revealed that MLAA-34 is most strongly expressed in U937 cells. Immunostaining indicated that MLAA-34 is localized in the cytoplasm and cell membrane. Furthermore, lentivirus-mediated overexpression of MLAA-34 in the U937 cell line led to significant suppression of apoptosis and increased the potential of tumorigenicity. Co-immunoprecipitation (Co-IP), shotgun and bioinformatic analysis identified 256 proteins and 225 of them were annotated by gene ontology categories. This analysis revealed 71 proteins involved in cell apoptosis or proliferation of biological processes and signaling pathways. Moreover, the effect of MLAA-34 apoptosis may be through interaction with the Ras, Wnt, calcium and chemokine signaling pathways and thirteen of the annotated proteins may interact with MLAA-34 and participate in carcinogenesis directly. This study provides a basis for a better understanding of the molecular mechanism and proteomics in the inhibition of apoptosis by MLAA-34 in U937 cells and indicates that MLAA-34 may be a potential candidate for the early diagnosis and therapeutic application of M5.