Abstract Bacterial sulfate reduction is significant for the karst development and pyrite formation within the Ordovician weathering crust in the Ordos Basin of China. Bacterial communities were studied to determine their potential geomicrobiological functioning by constructing 16S rRNA clone library for in situ samples. The results showed that 147 positive clones sequenced were divided into 23 operational taxonomic units (OTUs), 8 OTUs accouting for 80% of all the selected clones belonged to the genus Desulfosporosinus. Bacterial sulfate reduction has been demonstrated to take place in the Ordovician by the classical hydrogeological information together with the stable sulfur isotope analysis from both the pyrite in the weathering crust and the products of the laboratory experiments on the dissolution of sulfate rock. The H2S produced by bacterial sulfate reduction combined with iron to form pyrite, resulting in the development of hypogenic karst in the weathering crust. This process provided a reasonable interpretation for karst development in the vicinity of sulfur deposits in the Ordos Basin.