The dibenzothiophene (DBT) metabolic pathway in Mycobacterium strain G3, which is classified as a desulfurizing microorganism with the 4S pathway, was analyzed. 2-Hydroxybiphenyl (HBP), which is an end metabolite in the DBT desulfurization reaction, and 2-methoxybiphenyl (MBP) were found in the reaction mixture, and the methoxylation pathway from HBP to MBP was clarified. Although the substrate in the methoxylation reaction was HBP, there was no relationship between expression of the methoxylation activity and that of the desulfurization activity. Then, 4,6-dimethyl DBT, 4,6-diethyl DBT and benzo[b]naphtho[2,1-d]thiophene were metabolized to their methoxy forms via the desulfurization pathway. We established the methoxylation pathway in Mycobacterium G3.