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Purified porcine gastric mucin (PGM) is an alternative biomaterial to native mucin which displays multifunctional properties for exploring a wide range of biomedical applications. The present study evaluated the in vitro (RAW 264.7 macrophage cells) and in vivo (zebrafish embryos and larvae) bioactivities of PGM. The median lethal concentration (LC 50 ) of PGM was 197.9 µg/mL for embryos, while it was non-toxic to RAW 264.7 cells, even at 500 µg/mL. Following PGM exposure (100 µg/mL), a higher embryo hatching rate (59.9%) was observed at 48 h post fertilization, compared to the control (30.6%). Protective effects of PGM from pathogenic Aeromonas hydrophila were demonstrated by high larvae survival rates of 85.0% and 94.0% at 50 and 100 μg/mL of PGM exposure, respectively. Heat tolerance effect of PGM (50 and 100 µg/mL) on larvae (40 °C for 48 h) was confirmed by 75% and 100% of survival rates, respectively. Additionally, PGM reduced the A. hydrophila -induced reactive oxygen species (ROS) generation in larvae. The qRT-PCR results in PGM exposed larvae exhibited induction of immune-related genes ( tlr5a and tlr5b , myd88 , c-rel , il1β, tnf-α , il6, il10 , cxcl18b , ccl34a.4 , defbl1 , hamp , ctsd, muc2.1 , muc5.1 , muc5.2 , and muc5.3 ), stress response ( hsp70 , hsp90aa1.1 , and hsp90ab1 ), and antioxidant genes ( cat and sod1 ). Moreover, our results revealed that PGM involved in the regulation of transcriptional gene induction increases Hsp90 protein in the zebrafish larvae. Furthermore, upregulation of Il6, Il10, Tnfα, Ccl3, Defa-rs2, Defa21 and Camp and antioxidant genes ( Sod2 and Cat ) were observed in PGM-exposed RAW 264.7 cells. Overall findings confirmed the activation of immune responses, disease resistance against pathogenic bacteria, heat tolerance, and ROS-scavenging properties by PGM, which may provide insights into new applications for PGM as a multifunctional immunomodulator.