The Rho-kinases (ROCK) inhibitor Y-27632 has been shown to promote the growth of epidermal cells, however, its potential effects on human dermal fibroblasts (HDFs) need to be clarified. Here we show that prolonged treatment of HDFs with Y-27632 decreased their growth by inducing senescence, which was associated with induction of the senescence markers p16 and p21, and downmodulation of the ERK pathways. The senescent HDFs induced by Y-27632 acquired a cancer-associated-fibroblast (CAF)-like phenotype to promote squamous cell carcinoma (SCC) cell growth in vitro. Expression of a newly identified target of Y-27632 by RNA-seq, insulin growth factor binding protein 5 (IGFBP-5), was dramatically increased after 24 h of treatment with Y-27632. Adding recombinant IGFBP-5 protein to the culture medium produced similar phenotypes of HDFs as did treatment with Y-27632, and knockdown of IGFBP-5 blocked the Y-27632-induced senescence. Furthermore, Y-27632 induced the expression of an IGFBP-5 upstream gene, GATA4, and knockdown of GATA4 also reduced the Y-27632-induced senescence. In summary, these results demonstrate for the first time that Y-27632 promotes cellular senescence in primary HDFs by inducing the expression of IGFBP-5 and that prolonged treatment with Y-27632 potentially transforms primary HDFs into CAF-like cells.