Synthetic phenolic antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) has gained growing concerns due to relatively high concentrations in aquatic ecosystems. There are, however, significant knowledge gaps regarding its potential toxicity to aquatic organisms. In this study, zebrafish (Danio rerio) larvae were exposed to 0.01, 0.1, or 1 μM 2,4-DTBP for 6 d. Transcriptomic analysis of larvae revealed that biological processes related to anti-inflammatory function of macrophage M2 lineage were inhibited by 0.01 μM 2,4-DTBP. Decreases of transcripts related to the IL1B-MYD88-NF-κB pathway (i.e., il1b, il1rl1, myd88, irak4, irak1, traf6, ikbkg, nfkbia, nfkb) and protein levels of NF-κB in larvae intestine confirmed anti-inflammatory effects of 2,4-DTBP. Subsequently, larvae exposed to 2,4-DTBP were challenged with E. coli and showed higher survival rate, suggesting sustained activation of inflammation via LPS can be attenuated by 2,4-DTBP. Moreover, histological examination revealed that intestine barrier was compromised and there was an imbalance of intestine macrophage homeostasis. Food intake was also reduced following exposure to 0.1 and 1 μM 2,4-DTBP. In addition, a risk assessment revealed that 2,4-DTBP in surface water pose low to high ecological risks to aquatic organisms. Taken together, exposure to environmentally relevant concentrations of 2,4-DTBP could negatively affect immune response in zebrafish and may elicit ecological risk in fish population.