Animal studies and human clinical trials have shown that Lactobacillus can prevent or ameliorate inflammation in chronic colitis. However, molecular mechanisms for this effect have not been clearly elucidated. We hypothesize that lactobacilli are capable of downregulating pro‐inflammatory cytokine responses induced by the enteric microbiota. We investigated whether lactobacilli diminish production of tumour necrosis factor alpha (TNF‐α) by the murine macrophage line, RAW 264.7 gamma (NO–), and alter the TNF‐α/interleukin‐10 (IL‐10) balance, in vitro. When media conditioned by Lactobacillus rhamnosus GG (LGG) are co‐incubated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA), TNF‐α production is significantly inhibited compared to controls, whereas IL‐10 synthesis is unaffected. Interestingly, LGG‐conditioned media also decreases TNF‐α production of Helicobacter‐conditioned media‐activated peritoneal macrophages. Lactobacillus species may be capable of producing soluble molecules that inhibit TNF‐α production in activated macrophages. As overproduction of pro‐inflammatory cytokines, especially TNF‐α, is implicated in pathogenesis of chronic intestinal inflammation, enteric Lactobacillus‐mediated inhibition of pro‐inflammatory cytokine production and alteration of cytokine profiles may highlight an important immunomodulatory role for commensal bacteria in the gastrointestinal tract.