Denitrification for the treatment of nitrates in wastewater typically relies on organic electron donating substrates. However, for groundwater treatment, inorganic compounds such as elemental sulfur (S0) are being considered as alternative electron donors in order to overcome concerns that residual organics can cause biofouling. In this study, a packed-bed bioreactor supplied with S0:limestone granules (1:1, v/v) was started up utilizing a chemolithotrophic denitrifying enrichment culture in the form of biofilm granules that was pre-cultivated on thiosulfate. The granular enrichment culture enabled a rapid start-up of the bioreactor. A nearly complete removal of nitrate (7.3 mM) was NO3- attained by the bioreactor at nitrate loading rates of up to 21.6 mmol/(L(reactor)d). With lower influent concentrations (1.3 mM nitrate) comparable to those found in contaminated groundwater, high nitrate loads of 18.1 mmol/(L(reactor)d) were achieved with an average nitrate removal efficiency of 95.9%. The recovery of nitrogen as benign N2 gas was nearly stoichiometric. The concentration of undesirable products from S0-based denitrification such as nitrite and sulfide were low. Comparison of bioreactor results with batch kinetic studies revealed that denitrification rates were dependent on the surface area of the added S0. The surface area normalized denitrification rate was determined to be 26.4 mmol /(m2 S0 d).