With the application of wavelength division multiplexing, four-wave mixing (FWM) has an obvious influence on transmission systems. The effects of different channel intervals and numbers of channels on FWM performance in next generation Ethernet passive optical networks are discussed experimentally. When the incident signals near the zero-dispersion wavelength with a 200-GHz channel interval are transmitted over a 25-km fiber, new frequencies are generated, and more than 5-dB sensitivity loss is obtained. With the increase of channel spacing, the performance of FWM decreases significantly. The FWM crosstalk can even be ignored when the channel spacing is set at 800 GHz. In addition, the performance of FWM significantly increases when the number of channels is changed from 3 to 4, at which the power depletion is more severe.