The aberrant activation of autocrine motility factor receptor (AMFR) has been implicated in several types of human cancer. The present study aimed to elucidate the effect of AMFR on the regulation of proliferation in an acute monocytic leukemia cell line, THP‑1. THP‑1 cells were transfected with AMFR‑targeted small interfering (si)RNA and a plasmid encoding a truncated AMFR, AMFR‑C, (pcDNA3.1‑AMFR‑C). The mRNA and protein levels of AMFR and the downstream targets, rho‑associated, coiled‑coil containing protein kinase 2 (ROCK2), cyclin D1, and B‑cell lymphoma (Bcl)‑2, were measured using reverse transcription‑quantitatibe polymerase chain reaction and immunoblot analyses. The effects on cell cycle and apoptosis were investigated using flow cytometry. The present study successfully established the knockdown of AMFR and expression of AMFR‑C in the THP‑1 cells. Downregulation of AMFR induced cell cycle arrest at the G0/G1 phase, and increased apoptosis of the THP‑1 cells (all P<0.05). The AMFR siRNA increased the percentage of early apoptotic cells between 3.88±1.43 and 19.58±4.29% (P<0.05). The expression levels of ROCK2, cyclin D1 and Bcl‑2 were reduced by the downregulation of AMFR and enhanced by overexpression of AMFR‑C. In conclusion, AMFR appears to be crucial for the proliferation of the THP‑1 acute monocytic leukemia cell line. Therefore, AMFR may represent a potential target for the treatment of acute monocytic leukemia.