Naphthalene is one of the concentrated polycyclic aromatic hydrocarbons (PAHs) in cigarette mainstream smoke. Accumulated exposure to naphthalene causes various health problems such as irritation, gastrointestinal problems, neurological symptoms, renal problems, hematological disorders, and cancer. This study aimed to develop a biomass-based activated carbon from dried hemp (Cannabis sativa) stem for the removal of naphthalene from cigarette mainstream smoke. Activated carbon was prepared at an activation temperature of 350 to 550 °C using phosphoric acid as the activating agent. The elemental composition, porous texture, and surface functional groups of the activated carbon were determined by elemental analysis, nitrogen adsorption measurements, and temperature-programmed desorption (TPD). We found that the specific surface area and oxygen-containing functional groups of the carbon facilitate naphthalene adsorption where 77–96% of the naphthalene was removed from cigarette mainstream smoke. In addition, a computational investigation with a semiempirical PM7 calculation has provided the adsorption energies of naphthalene on oxygen-containing functional groups. As expected, the carboxyl group showed high efficiency in naphthalene adsorption, which is consistent with the experimental results.