Under physiological conditions, the recruitment of macrophage to sites of infection is an important physiological process in host defenses. It has been reported that Connexin43 (Cx43), the most ubiquitous gap junction protein subunit, may be involved in inflammation response of immune cells, including macrophages. However, the underlying molecular mechanisms remain uncertain. In this study, RAW264.7 cell line and thioglycolate-elicited peritoneal macrophages (TGEMs) were used and we observed that lipopolysaccharide (LPS)-contained conditional medium (CM) upregulated Cx43 protein and mRNA expression. Besides, LPS treatment also caused the increase of Cx43 in a dose- and time-dependent manner with up-regulated phosphorylation of AKT, mTOR and SGK. Additionally, overexpression or silencing of Cx43 led to the enhancement or inhibition of CM-induced TGEMs migration, respectively. Mechanistically, the inhibitory effect by knockdown of Cx43 was mediated by the inhibition of nitric oxide (NO) production, inducible nitric-oxide synthase (iNOS), focal adhesion kinase (FAK) and Src pathways. Inhibition of CM-induced TGEMs migration with downregulation of NO production, FAK and Src were also observed by 1400W, an iNOS inhibitor, suggesting the involvement of iNOS-Src-FAK pathway in Cx43-mediated CM-stimulated macrophage migration. On the other hand, the treatment of serum, a SGK activator, upregulated Cx43 expression; and both Cx43 expression and CM-induced TGEMs migration were suppressed either by knocking down SGK gene or by the blockade of the SGK pathway. Besides, LPS-induced SGK activation was abrogated not by rapamycin but by Torin2, while LPS-induced Cx43 was reduced by both inhibitors. Additionally, methylprednisolone or tacrolimus (FK506), common choices following solid organ transplantations, decreased LPS-induced Cx43 expression or SGK activation in TGEMs, indicating the link between the usages of tacrolimus or glucocorticoid in transplantation medicine and impaired macrophage migration via Cx43-iNOS-Src-FAK pathway. Collectively, our study reveals the function of Cx43 in CM or LPS-stimulated macrophage, reaffirming the implication of Cx43 in the host response to microbial infection.