Rumen cannulation is a widely employed technique in ruminant nutrition research. However, the gap between skin and rumen cannula can cause leakage of fermentation gases and influx of atmospheric air, which may adversely affect the anaerobic environment in the rumen. The present study was designed to investigate the effects of rumen cannulation on headspace gases, dissolved gases, fermentation end products, and methanogen community in the rumen of dairy cows. Eight Holstein cows were used in the experiment. Four cows were surgically fitted with rumen cannulas, whereas the other 4 intact cows were used as control. Rumen cannulation decreased gaseous hydrogen and methane concentrations, dissolved carbon dioxide concentration, and relative abundances of Methanosphaera, and increased the saturation factor of dissolved hydrogen and dissolved methane, dissolved methane concentration, volatile fatty acid concentration, 16S ribosomal RNA gene copies of methanogens, and Simpson index of methanogen community. In summary, rumen cannulation causes a reduction in headspace gaseous hydrogen and gaseous methane, which may not decrease dissolved gas concentrations due to an increase in saturation factors. Furthermore, rumen cannulation alters methanogen community with increased methanogen population and decreased relative abundances of Methanosphaera.