The strength and source of anthropogenic sulfur (S) deposition is an important area of research in both environmental and ecological disciplines. Here S concentration, stable isotope (δ³⁴S) and photosynthetic pigments analyses were performed on epilithic mosses at Guiyang area (SW China) for investigating the distribution, origin and effect of urban-derived S at the urban–rural scale. Based on the variation of moss S, anthropogenic S was estimated to account for about 52% of total S in urban mosses and 35% in rural mosses within 30 km. The deposition of urban-derived S was biologically determined within 57 km from the urban center, but only 22% (about 24.4 × 10⁶ kg-S annually) reached over 30 km, with 78% (about 85.9 × 10⁶ kg-S) deposited within 30 km. δ³⁴S_moss signatures suggested the major source of anthropogenic S was still from coal combustion, and comparable δ³⁴S_moss values along the urban–rural transect suggested the inputs of urban-derived S into rural ecosystems. Unexpectedly, moss photosynthetic pigments did not show a decrease with S deposition, but express higher concentrations in the urban than in the rural. In contrast, correlations between moss photosynthetic pigments, and tissue nitrogen (N) and δ¹⁵N demonstrated a fertilizing effect of elevated N deposition on moss photosynthesis, which might buffer or offset the negative effect of S deposition on urban mosses. Collective evidences suggest that S% and δ³⁴S in epilithic mosses provided useful information for determining anthropogenic S deposition on the urban–rural scale, but moss photosynthetic pigments may not be applicable for reflecting S loading under high N deposition.