Ponds are widely distributed in rice-based agricultural watersheds, particularly in southern China, and may play an important role in nitrate () removal. However, the denitrification rate of pond sediment, measured using the acetylene (C2H2) inhibition technique, indicated that the amount of nitrogen removed by denitrification accounted for <1% of the total nitrogen applied. The present study was undertaken to determine the effects of sediment depth and temperature on denitrification of pond sediment using the C2H2 inhibition technique. The highest denitrification potential was found in the upper 5 cm of sediment, but this only accounted for approximately 34% of the total denitrification of the upper 0–30 cm of sediment, suggesting that sediment denitrification potential would be underestimated if only the upper 5 cm of sediment was measured. The denitrification potential was low and showed a weak response over a temperature range of 6–18°C, whereas denitrification increased significantly from 18 to 30°C, indicating that denitrification may play an important role in the removal of in warm seasons. A comparison of the disappearance and C2H2 inhibition methods showed that they were significantly (P < 0.01) and positively correlated. However, the C2H2 inhibition method gave only approximately 25% of the values determined by the disappearance method.