Plants metabolize polychlorinated biphenyls (PCBs) into hydroxylated derivatives (OH-PCBs), which are sometimes more toxic than the parent PCBs. The objective of this research was to compare the toxicity of a suite of PCBs and OH-PCBs toward the model plant, Arabidopsis thaliana. While parent PCBs and higher-chlorinated OH-PCBs exhibited a low or nondetectable toxicity, lower-chlorinated OH-PCBs significantly inhibited the germination rate and plant growth, with inhibition concentration 50% (IC50) ranging from 1.6 to 12.0 mg L−1. The transcriptomic response of A. thaliana to 2,5-dichlorobiphenyl (2,5-DCB), and its OH metabolite, 4′-OH-2,5-DCB, was then examined using whole-genome expression microarrays (Affymetrix). Exposure to 2,5-DCB and 4′-OH-2,5-DCB resulted in different expression patterns, with the former leading to enrichment of genes involved in response to toxic stress and detoxification functions. Exposure to 2,5-DCB induced multiple xenobiotic response genes, such as cytochrome P-450 and glutathione S-transferases, potentially involved in the PCB metabolism. On the contrary, exposure to both compounds resulted in the down-regulation of genes involved in stresses not directly related to toxicity. Unlike its OH derivative, 2,5-DCB was shown to induce a transcriptomic profile similar to plant safeners, which are nontoxic chemicals stimulating detoxification pathways in plants. The differentiated induction of detoxification enzymes by 2,5-DCB may explain its lower phytotoxicity compared to 4′-OH-2,5-DCB.