Carbon stable isotope ratios (δ¹³C) are widely used to trace resource transfer pathway yet δ¹³C variation of freshwater autotrophs has not been fully understood. We analyzed data from 42 published studies supplemented with some unpublished data to show the determinants of lotic periphyton δ¹³C. At large spatial scales, we observed broad differences in periphyton δ¹³C among biomes and consistent longitudinal variation related to watershed area. Longitudinal increases in δ¹³C indicated the importance of in-stream processes on lotic carbon cycles and autotroph δ¹³C variation. At local spatial scales, periphyton δ¹³C was related negatively to canopy cover and water current velocity and positively to chlorophyll a density. Autotroph δ¹³C varied among taxonomic groups. Cyanobacteria and red algae had significantly higher and lower δ¹³C than other taxa, respectively. A hierarchical model across spatial scales showed that local controls for periphyton δ¹³C were nested by regional controls, which suggested that productivity and CO₂ availability determine δ¹³C. Overall our results revealed general patterns of periphyton δ¹³C and provided improved information for study design and use of δ¹³C in isotopic mixing models in lotic food web studies.