Two limiting factors of microbial electrochemical denitrification (MED) are the abundance and efficiency of the functional microorganisms. To supply these microorganisms, MED systems are inoculated with denitrifying sludge, but such method has much room for improvement. This study compared MED inoculated with autotrophic denitrifying inoculum (ADI) versus with heterotrophic denitrifying inoculum (HDI). ADI exhibited electroactivity for 50% less of time than HDI. The denitrification efficiency of the ADI biocathode was 42% higher than that of the HDI biocathode. The HDI biocathode had high levels of polysaccharides while the ADI biocathode was rich in proteins, suggesting that two biocathodes may achieve MED but via different pathways. Microbial communities of two biocathodes indicated MED of HDI biocathode may rely on interspecies electron transfer, whereas sulfur bacteria of ADI biocathode take electrons directly from the cathode to achieve MED. Utilizing autotrophic sulfur-oxidizing denitrifiers, this study offers a strategy for enhancing MED.