Numerous studies indicate that the interaction between cancer-associated fibroblasts (CAFs) and tumors is manifested in the entire process of colorectal cancer (CRC) cell development, in which TGF-β1 plays a key role and has a significant effect on promoting the activation of CAFs. However, there are few studies on the mechanisms involved in the activation of CAFs by TGF-β1 to produce an influence on tumor cells. TGF-β1 was added to CAFs for further culture, and the expression of α-SMA was significantly enhanced as shown by immunofluorescence assay. Western blot analysis was performed, and the results showed that TGF-β1 promoted expression of PGP9.5 in a time-dependent manner. After siRNA was used to inhibit the expression of Smad2 or Smad3, the TGF-β1-induced PGP9.5 expression in CAFs was obviously suppressed. In addition, TGF-β1 was also found to promote the expression of PGP9.5 through the ERK1/2 and PI3K pathways. CAFs were cultured on the upper layer of a Transwell plate and TGF-β1 was added. Simultaneously, CRC cells were cultured on the lower layer. The biological behaviors of the cancer cells were detected. According to the results, TGF-β1 promoted the proliferation and invasion of CRC cells and inhibited their apoptosis while activating CAFs. This effect was achieved by induction of the expression of PGP9.5. However, when PGP9.5 was inhibited, the impact of TGF-β1 on tumor cells after activation of CAFs was not fully blocked. Therefore, TGF-β1 can promote PGP9.5 expression in CAFs to facilitate the growth of cancer cells. This finding aids in the identification of new targets for treating CRC.