Musty and earthy substances represented by 2-methylisoborneol (2-MIB) are important pollutants not only affecting the water quality but also threatening the security of drinking water sources in lakes and reservoirs. In this study, we investigated the spatial and seasonal distributions of 2-MIB in waters of Lake Yangcheng, a large eutrophic shallow lake in China. Driving factors affecting the production and accumulation of 2-MIB were further analyzed. The results showed that 2-MIB concentrations in Lake Yangcheng ranged from 0.4 to 940.6 ng/L, with a high annual average concentration of 35.6 ng/L. Distinct seasonal and spatial variations of 2-MIB were found in the lake waters. Specifically, 2-MIB concentrations were highest in summer, followed by spring and autumn, and lowest in winter, whereas 2-MIB concentrations generally exceeded the odor threshold (< 10 ng/L) in the first three seasons. Spatially, 2-MIB concentrations were highest in the eastern lake, and followed by the central and the western lake. The environmental conditions of a higher water temperature and lower TN:TP ratio (TN:TP < 29:1) were more favorable to the production and accumulation of 2-MIB in Lake Yangcheng. The algal abundance analysis and correlation analysis showed that cyanobacteria species including Microcystis spp. and Phormidium spp. were closely related to the production of 2-MIB and were considered as the main driving factors of the accumulation of 2-MIB in Lake Yangcheng. Therefore, controlling the eutrophication level and algal blooms are of great importance to alleviate 2-MIB pollution in lake waters.