Abstract: Toll-like receptor (TLR) family is an evolutionarily conserved pathogen recognition receptor, playing an important role in detecting and defending against microbial pathogens. To investigate the role of the HcTLR1 gene in the antimicrobial response of Hyriopsis cumingii, the full-length cDNA sequence of the HcTLR1 gene was cloned using rapid-amplification of cDNA ends (RACE) technology; real-time fluorescence quantitative PCR analysis was employed to compare the expression levels of the HcTLR1 gene in various tissues of H. cumingii challenged with different stimuli; double-stranded RNA interference technology was used to analyze the changes in the MyD88-dependent pathway and related immune genes after the knockdown of the gene. The results show that the open reading frame (ORF) of the HcTLR1 gene was 3 687 bp, encoding 1 228 amino acids. The predicted structure of the HcTLR1 protein contained multiple Leucine-rich repeat domains, a transmembrane domain and an intracellular Toll/interleukin-1 receptor. Furthermore, the mRNA expression of HcTLR1 gene was highest in hemocytes and exhibited significant changes in response to Aeromonas veronii GL1 and pathogen-associated molecular patterns stimulation at different time points. Moreover, the knockdown of the HcTLR1 gene significantly reduced the expression levels of genes in MyD88-related pathway, antibacterial peptides, lysozyme, defensins, lactoferrin, LPS1-binding protein/bactericidal permeability-increasing protein 2, and interleukin 17 stimulated by A. veronii GL1. In conclusion, it is suggested that HcTLR1 activates MyD88-dependent signaling pathways in H. cumingii during microbial infection and promotes resistance mechanisms in hemocytes.