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BACKGROUND In ruminants, enteric CH4 represents a major energy loss for the host and is a potent greenhouse gas that contributes to climate change. Previous studies have shown that humic substances (HS) may have beneficial effects on livestock nutrition. The present study investigated the effects of HS on in vitro CH4 production and rumen fermentation. RESULTS Total gas production was linearly increased with increasing HS after 12 h of incubation, although it was unaffected after 24 and 48 h. Increasing HS linearly decreased CH4 at all time points. Increasing HS linearly decreased NH3-N concentration and the molar proportion of acetate at 12 h, whereas the efficiency of microbial protein (MP) production and total dry matter digestibility (TDMD) linearly increased, with starch digestion (SD) responding quadratically. After 48 h, HS linearly increased MP and TDMD, with neutral detergent fibre digestibility responding quadratically. CONCLUSION Inclusion of HS effectively reduced CH4 production and increased substrate disappearance and the efficiency of microbial protein synthesis in vitro. However, its effect on in vivo CH4 production, rumen fermentation and ruminant production requires further investigation. (c) 2018 Society of Chemical Industry