Humic acid inhibits methane production by competing with the methanogenic process for electrons and is a natural strategy for reducing greenhouse gas emissions from lakes. There is still a lack of systematic understanding of the structural characteristics of humic acid in different lake habitat types and its impact on the methanogenesis process. This study aimed to systematically analyze the structural characteristics of humic acid (HA) in sediments from different regions of Lake Taihu and its effects on methanogenesis. The sampling area covers East Taihu Lake, Meiliang Bay, the central lake area and Dapukou, and surface sediments were collected for quantitative analysis. The content, structural characteristics and redox properties of HA (including electron accepting capacity (EAC) and electron donating capacity (EDC)) were characterized by Fourier transform infrared spectroscopy, spectrophotometry, elemental analysis, electron transfer capacity measurement and quantitative measurement of quinone groups. The results showed significant spatial differences in HA content and its redox properties among the four regions of Lake Taihu. The HA content in sediments from grass-type lakes (such as East Lake Taihu) was the highest, while the EAC of HA in sediments from algae-type lakes was the strongest. Correlation analysis showed that the EAC of HA was significantly negatively correlated with the carbon/nitrogen ratio of sediments and significantly positively correlated with the aromatic condensation degree of HA. To further clarify the effect of Lake Taihu HA on the methanogenesis process, this study explored the effect of HA from different EAC on the methanogenesis process in a methanogen enrichment system and monitored the methanogenesis process by measuring the methane accumulation and the relative activity of coenzyme F420. The results showed that as the EAC of HA increased, the inhibitory effect of HA on the methanogenesis process became more significant and the relative activity of coenzyme F420 became lower. This finding suggests that EAC from lake HA inhibits methane production by suppressing the activity of methanogens. Future studies should further focus on the effects of different matrices on the formation mechanism of lake HA and the key factors affecting the EAC of HA to fully understand the potential role of lake HA in climate change regulation.