The global spillover of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus into mammalian hosts has become a public health concern as well as to the equine industry. However, previous reports on H5N1 natural infection and the presence of specific antibodies in horses remain limited. Therefore, we conducted a serosurveillance study to assess the prevalence of H5N1 antibodies collected from 400 horses in Japan. Samples were screened using competitive ELISA (cELISA) and hemagglutination inhibition (HI) tests against H5-recombinant protein and H5N1 viral antigen, respectively. Of the 400 samples, 399 (99.75%) were negative by cELISA, while one sample (0.25%) yielded a doubtful result. However, all samples showed HI titers of <8, confirming the absence of H5N1 antibodies. These findings indicate that the current risk of H5N1 infection among horses in Japan remains limited. Nonetheless, continuous global surveillance is warranted to monitor the potential risk of H5N1 spillover into equine populations.