Short-chain fatty acids (SCFA) produced from dietary non-digestible carbohydrate fermentation have metabolic effects in skeletal muscle; however, their effect on inflammatory mediator production is unknown. In this study, L6 myotubes were cultured with individual SCFA (acetate, propionate, and butyrate) at 0.5 mM and 2.5 mM ± 10 ng/mL lipopolysaccharide (LPS) or ± 500 µM palmitic acid (PA) for 24 h. In response to LPS, only butyrate had an effect at the lower concentration (0.5 mM), whereas at the higher concentration (2.5 mM) both propionate and butyrate reduced MCP-1, MIP-1α, and RANTES secretion (p < 0.05), and only butyrate reduced IL-6 secretion and intracellular protein levels of phospho-STAT3 (p < 0.05). In response to PA, 0.5 mM butyrate reduced protein expression of phospho-NFκB p65 and the secretion of IL-6, MIP-1α, and MCP-1, whereas all three SCFA reduced RANTES secretion (p < 0.05). At the 2.5 mM SCFA concentration combined with PA stimulation, all three SCFA reduced intracellular protein expression of phospho-NFκB p65 and phospho-STAT3 and secreted protein levels of MCP-1, IL-6, and RANTES, whereas only butyrate reduced secretion of MIP-1α (p < 0.05). Thus, SCFA exhibit differential effects on inflammatory mediator expression in response to LPS and PA stimulation, which has implications for their individual impacts on inflammation-mediated skeletal muscle dysfunction.