Dibenzothiophene (DBT) is an organic sulfur compound which remains in oil after hydrodesulfurization (HDS) process and can be removed by biodesulfurization (BDS). A new strain of <em>Paenibacillus validus</em> (strain PD2) was isolated from oil contaminated soils that is able to desulfurize DBT. HPLC analysis and Gibb’s assay showed that this strain was capable to convert DBT to 2-Hydroxybiphenyl (2-HBP) as final product. The presence of <em>dsz</em>C gene confirmed that DBT desulfurization occurred through the 4S pathway. Maximum growth and the highest induction in dsz operon obtained in the presence of dimethyl sulfoxide (DMSO) as sole sulfur source. DBT concentration, temperature and pH were optimized statistically for growing and resting cells by using Response Surface Methodology (RSM). All parameters in growing cells had a significant effect on 2-HBP production during BDS of DBT by <em>P. validus</em> PD2, although in resting cells temperature in range of 20–40°C was not a significant factor. Maximum BDS for growing cells was obtained at 0.41 mM DBT concentration, pH 6.92 and temperature 31.23°C. For resting cells, optimum pH, temperature and DBT concentration were 6.62, 27.73°C and 7.86 mM respectively. The results of this study showed that high concentrations of DBT could be desulfurized by <em>P. validus</em> strain PD2 in model-oil. Thus, the isolated strain could be introduced as a proper candidate for biodesulfurization of organic sulfur in the oil industry.