Highway runoff is a known source of pollution; however, its ability to sequester carbon is less well understood. In this study, we examined the pollution, sources, and carbon sequestration of highway runoff in karst areas of Guangxi, China. Runoff samples were collected from different locations during the rainy season. Suspended solids, CODMn, NH3–N, and Mn were identified as the primary pollutants. Factor analysis revealed five main sources of highway runoff: traffic, construction, carbonate weathering, rainfall combined with artificial fertilizers, and artificial fertilizers alone. Traffic and carbonate weathering emerged as the predominant factors in the Yaji section of the Guilin Ring Expressway (YJ) and adjacent to the Baizhujing Reservoir (BZJ), while construction and carbonate weather were the primary factors in Xincheng County (XC). The carbon sequestration capacity of highway runoff was the highest in XC, followed by BZJ and YJ. YJ and BZJ accounted for 15-21% of that observed in a karst spring in the same region, revealing a 3.44-4.64–fold increase compared to silicate basins. Thus, despite being a source of pollution, the function of highway runoff as a carbon sink may be key to emission reduction efforts.