The decomposition of polyethylene (PE), an extremely recalcitrant synthetic polymer, using microorganisms is an ideal and sustainable method for future PE biotreatment. We isolated a set of PE-biodegrading Bacillus species from a landfill site. Among them, Bacillus thuringiensis JNU01 exhibited the highest cell growth rate in PE media, which means it effectively decomposed PE to use in the metabolic pathway as a sole carbon source. B. thuringiensis JNU01-treated PE showed new chemical functional groups such as hydroxyl, carboxyl, and amide groups in the inert hydrocarbon. Scanning electron microscopy revealed considerable physical damage on the surface of the PE film after treatment with B. thuringiensis JNU01. Furthermore, various alkane derivatives obtained from PE were characterized using gas chromatography–mass spectrometry. On the contrary, an increase in the mRNA transcriptional levels of B. thuringiensis JNU01 in the presence of PE suggests that a CYP102A5 variant (CYP102A5.v1) is involved in PE biodegradation. Finally, we confirmed that purified CYP102A5.v1 catalyzes the hydroxylation of PE by a NADPH oxidation assay and Fourier transform infrared analysis. These results show that B. thuringiensis JNU01 is a potential PE decomposer and suggest that CYP102A5.v1 can be a trigger biocatalyst for hydroxylation of PE.