SummaryHeterotrimeric Gi proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS‐induced, heat‐killed SA‐induced and heat‐killed GBS‐induced cytokine and chemokine production in peritoneal macrophages from wild‐type (WT), Gαi2–/– or Gαi1/3–/– mice were investigated. LPS induced production of tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), IL‐10 and interferon‐γ‐inducible protein‐10 (IP‐10); SA induced TNF‐α, and IL‐1β production; and GBS induced TNF‐α, IL‐6, IL‐1β, macrophage inflammatory protein‐1α (MIP‐1α) and keratinocyte chemoattract (KC) production were all decreased (P < 0·05) in Gαi2–/– or Gαi1/3–/– mice compared with WT mice. In contrast to the role of Gi proteins as a positive regulator of mediators, LPS‐induced production of MIP‐1α and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) were increased in macrophages from Gαi1/3–/– mice, and SA‐induced MIP‐1α production was increased in both groups of Gαi protein‐depleted mice. LPS‐induced production of KC and IL‐1β, SA‐induced production of GM‐CSF, KC and IP‐10, and GBS‐induced production of IL‐10, GM‐CSF and IP‐10 were unchanged in macrophages from Gαi2–/– or Gαi1/3–/– mice compared with WT mice. These data suggest that Gi2 and Gi1/3 proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram‐positive microbial stimuli.