Platelet‐activating factor (PAF) is an important lipid mediator of anaphylaxis and therefore can be an anti‐anaphylactic agent target. Recently, we reported that several synthetic biotinylated peptides containing a Tyr‐Lys‐Asp‐Gly sequence markedly inhibited the bioactivities of PAF in vitro and in vivo; it also inhibited anaphylactic reactions such as hypothermia, hypotension, and vascular permeability in vivo. Here, we report the anti‐anaphylactic effects of three biotinylated heptapeptides (peptide 1: H‐Lys(biotinyl)‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp‐OH, peptide 2: H‐D‐Lys(biotinyl)‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp‐OH, and peptide 3: H‐D‐Lys(biotinyl)‐Trp‐Tyr‐Lys‐Asp‐Gly‐D‐Asp‐OH). The experiment using tryptophan fluorescence spectroscopy showed that the interaction of peptides 2 and 3 with PAF was larger than that of peptide 1. Experiments using a rat model of hind paw edema showed that peptides 1, 3, and 2 inhibited PAF‐induced edema by 67.9%, 69.3%, and 79.3%, respectively. In a mouse model of anaphylaxis, both peptides 2 and 3 showed inhibitory effects on anaphylactic hypothermia, whereas peptide 1 did not. Furthermore, experiments involving in vitro rat plasma stability of peptides showed that both peptides 3 and 2 were more stable in plasma compared to peptide 1 (84.0%, 51.8%, and 0%, remained after 6 h, respectively). Our results suggest that both peptides 2 and 3 may show systemic and local inhibitory effects as anti‐anaphylactic agents targeting PAF.