Polychlorinated biphenyls (PCBs) are highly persistent and ubiquitously distributed environmental pollutants. Based on their chemical structure, PCBs are classified into non-ortho-substituted and ortho-substituted congeners. Ortho-substituted PCBs are structurally similar to dioxin and their toxic effects and mode of action are well established. In contrast, very little is known about the effects of non-ortho-substituted PCBs, particularly during early development. The objective of this study is to investigate the effects of exposure to an environmentally prominent ortho-substituted PCB (2,2’,4,4’,5,5’-hexachlorobiphenyl; PCB153) on zebrafish embryos. We exposed zebrafish embryos to either DMSO or three different concentrations of PCB153 starting from 4 hours post-fertilization (hpf) to 120 hpf. We quantified gross morphological changes, behavioral phenotypes, gene expression changes and circadian behavior in the larvae. There were no developmental defects during the exposure period, but starting at 7 dpf, we observed spinal deformity in the 10 μM PCB153 treated group. A total of 633, 2227, and 3378 differentially expressed genes were observed in 0.1, 1 and 10 μM PCB153 treated embryos, respectively. Of these, 301 genes were common to all treatment groups. KEGG pathway analysis revealed enrichment of genes related to circadian rhythm, FoxO signaling and insulin resistance pathways. Behavioral analysis revealed that PCB153 exposure significantly alters circadian behavior. Disruption of circadian rhythms has been associated with the development of metabolic and neurological diseases. Thus, understanding the mechanisms of action on environmental chemicals in disrupting circadian regulation of metabolism is essential for preventing or mitigating chemical-induced metabolic disease.