The myeloid-associated Ig-like receptor family (CD300) consists of nine activating or inhibitory cell surface receptors preferentially expressed on myeloid cells and are encoded by the genes in a small cluster on mouse chromosome 11. One of the receptors, CD300LF (MAIR-V), has a long cytoplasmic tail containing two consensus ITIMs and an immunoreceptor tyrosine-based switching motif, suggesting that CD300LF regulates the activation of myeloid cells. However, the functional characteristics of this receptor are still incompletely understood. In this study, we demonstrate that cross-linking CD300LF with anti-CD300LF mAb induced cell death in peritoneal macrophages as well as in several transfectants expressing CD300LF. CD300LF-mediated cell death was dependent on the cytoplasmic region but did not require an ITIM or immunoreceptor tyrosine-based switching motif. Scanning electron microscopy revealed a loss of blebs from the surface of the dead cells mediated by CD300LF, a morphological feature similar to that observed in apoptotic cells. However, CD300LF-mediated cell death was not inhibited by a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or autophagy inhibitors, 3-methyladenine or N-acetyl-L-cystein. Moreover, the splicing isoform of a transcription factor, X-box binding protein-1, which is produced in dead cells as a response to endoplasmic reticulum stress, was not detected. Together, these results indicate that CD300LF mediates caspase and endoplasmic reticulum stress-independent cell death by a novel mechanism.