Recently, we demonstrated that Rac1 upregulation is involved in augmented bronchial smooth muscle (BSM) contractions of antigen-challenged mice. However, change in G protein-coupled receptor (GPCR)-induced Rac1 activation remains unknown in BSMs of repeatedly antigen-challenged (Chal.) mice. We here examined carbachol (CCh)-induced Rac1 activation in BSMs of Chal. mice. Gene expression levels of both Rac1 and Rac-guanine nucleotide exchange factors (GEFs), such as Tiam1 and Trio, were increased in BSMs of Chal. mice. Furthermore, CCh-induced Rac1 activation was inhibited by pretreatment with Rac1-GEF inhibitor NSC23766 and Rac1 inhibitor EHT1864 in BSMs of sensitized-control (S.C.) and Chal. mice. Compared with S.C. mice, CCh-induced Rac1 activation was increased in BSMs of Chal. mice. In conclusion, we reported that increased CCh-induced Rac1 activation via Tiam1 and Trio upregulation, in addition to upregulate Rac1, may be involved in increased CCh-induced BSM contractions in Chal. mice.