Recent crystallographic studies revealed that G‐protein‐coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8, at the proximal region of the C‐terminal tail. However, the significance of helix 8 in GPCR functions and signaling is not fully understood. Helix 8 mutants of leukotriene B₄ receptor type 1 (BLT1) exhibit prolonged activation after ligand stimulation, suggesting some regulatory roles of helix 8 in GPCR signaling. Here, we report the inhibitory role of BLT1 helix 8 on ligand‐dependent internalization using BLT1 and platelet‐activating factor receptor as model GPCRs. Mutating the dileucine motif in helix 8 of BLT1 to alanines (BLT1 LLAA) enhanced LTB₄‐dependent internalization of BLT1, whereas wild‐type (WT) BLT1 exhibited minimal internalization. Mutational studies revealed that phosphorylation of 5 serine/threonine residues between amino acids 308 and 319 of BLT1 was responsible for enhanced ligand‐dependent internalization of BLT1 LLAA. BLT1 LLAA showed enhanced basal and ligand‐dependent phosphorylation compared to WT BLT1. Taken together, helix 8 of BLT1 inhibits receptor internalization by suppressing the excessive phosphorylation of the C‐terminal tail.—Aratake, Y., Okuno, T., Matsunobu, T., Saeki, K., Takayanagi, R., Furuya, S., Yokomizo, T. Helix 8 of leukotriene B₄ receptor 1 inhibits ligand‐induced internalization. FASEB J. 26, 4068–4078 (2012). www.fasebj.org