Various molecules have been reported to be involved in the induction of HL-60 cell differentiation by all-trans retinoic acid (ATRA). Notably, the transfection of an antisense oligonucleotide targeting proteinase 3 (PRTN3, formerly known as myeloblastin) into HL-60 cells induces differentiation, though the underlying mechanism remains unclear. Here we found that the levels of secreted PRTN3 were significantly reduced when differentiation was initiated by ATRA treatment, suggesting that secreted PRTN3 inhibits HL-60 cell differentiation. The addition of anti-PRTN3 antibody to the culture medium of HL-60 cells promoted their differentiation. Conversely, when ATRA-treated HL-60 cells were cultured in conditioned media prepared from HEK293 cells transfected with the preproPRTN3 plasmid, differentiation was significantly suppressed. Moreover, conditioned media from HEK293 cells transfected with an inactive PRTN3 mutant (S203G) also suppressed HL-60 cell differentiation. These results suggest that PRTN3 secreted from HL-60 cells has an autocrine inhibitory effect on differentiation, independent of its enzymatic activity.