Anaerobic biodegradation of hexazinone was investigated in four sediments (L1, L2, Y1 and Y2). Results showed that the L2 sediment had the highest biodegradation potential among four sediments. However, the Y1 and Y2 sediments had no capacity to biodegrade hexazinone. Sediments with rich total organic carbon, long-term contamination history by hexazinone and neutral pH may have a high biodegradation potential because the former two factors can induce the growth of microorganisms responsible for biodegradation and the third factor can offer suitable conditions for biodegradation. The addition of sulfate or nitrate as electron acceptors enhanced hexazinone degradation. As expected, the addition of electron donors (lactate, acetate or pyruvate) substantially inhibited the degradation. In natural environmental conditions, the effect of intermediate A [3-(4-hydroxycyclohexyl)-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H, 3H)dione] on anaerobic hexazinone degradation was negligible because of its low level.