In order to explore the mechanism of innate immunity in Onchidium reevesii under hypoxia stress, the cDNA sequence of Toll-like receptor 4 (TLR4) gene was cloned by RACE-PCR, and bioinformatics analysis was conducted. In addtion, the expression changes of TLR4, the activity changes of hemocyte, lysozyme (LSZ), superoxide dismutase (SOD) and alkaline phosphatase (AKP) under hypoxia stress were determined. The results showed that the full length of TLR4 cDNA was 3,605 bp, the length of open reading frame was 2,817 bp, and it encoded a total of 956 amino acids. The phylogenetic tree results suggest that the TLR4 gene was the closest to the TLR4 gene of Biomphalaria glabrata. The results of qRT-PCR indicated that the TLR4 gene was expressed in all tissues, and the relative expression in the hepatopancreas was the highest. The expression of each tissue increased significantly under hypoxia stress and the expression of ganglion reached its peak earlier than the other tissues at 4 h. In addition, hemocyte viability, LSZ activity and AKP activity decreased at first and then increased, while SOD activity increased at first, then decreased and finally increased, which was more sensitive to hypoxia stress than the others. The results provide a data base for illustrating TLR4 biological functions and a theoretical foundation for investigation the mechanism of the immune system in intertidal macrobenthos under hypoxia stress.