The effects of sulfur on the fermentation characteristics of corn distiller's dried grains with solubles (DDGS) in in vitro culture were investigated. Samples (DDGS) were analyzed for nutrient values and then two independent in vitro experiments were conducted to study the effects of various sulfur sources (Na 2 S, Na 2 S 2 O 4 , Na 2 SO 3 , and Na 2 SO 4 ) and different sulfur levels (0.346, 0.692, and 1.038%) on the fermentation characteristics of DDGS. Based on sampling and chemical composition analysis, there existed a great variation in the concentrations of sulfur and proximate nutrients of DDGS. In Experiment 1, sulfur source showed a significant (P < 0.01) effect on the gas production parameters (asymptotic gas production (b) and gas production rate (c)) and gas production of DDGS -sulfur from Na 2 SO 4 and Na 2 S produced more (P < 0.01) gas within 48 h with a faster gas production rate as well as higher digestibilities (dry matter degradability and organic matter digestibility) and more energy supplies (metabolizable energy), net energy for maintenance and gain, and net energy for gain than sulfur from Na 2 SO 3 and Na 2 S 2 O 4 . Neither ammonia-nitrogen (NH 3 -N) concentration nor volatile fatty acids (VFA) profile (total VFA and individual VFA proportion) were affected by sulfur source (P > 0.05). In Experiment 2, no significant (P > 0.05) effect on the fermentation characteristics of DDGS with increasing sulfur content was found. The collective findings suggest that regular chemical analyses are necessary to make full use of DDGS, and that the valence state of sulfur in DDGS exerts an effect on its in vitro fermentation characteristics and there appears no dose-related effect of sulfur on the fermentation of DDGS in a short-term in vitro culture.Keywords: DDGS; rumen fermentation; byproduct; feeding value List of abbreviations: DDGS = distiller's dried grains with solubles, GP = gas production, DM = dry matter, EE = ether extract, NDF = neutral detergent fibre, ADF = acid detergent fibre, ADL = acid detergent lignin, Ca = calcium, P = phosphorus, CP = crude protein, NDICP = neutral detergent insoluble crude protein, ADICP = acid detergent insoluble crude protein, VFA = volatile fatty acids, MCP = microbial crude protein production, SCFA = short chain fatty acid, DDG = distiller's dried grains, DDS = distiller's dried solubles, SRB = sulfate-reducing bacteria, OM = organic matter, OMD = organic matter digestibility, DMD = dry matter degradability, NEm = net energy for maintenance, NEg = net energy for gain, ME = metabolizable energy, PF = partitioning factor