BACKGROUND/AIM: The tumor microenvironment greatly influences cancer occurrence, progression, and treatment resistance, making it a key target alongside cancer cells. In squamous cell carcinoma, the invasive front is crucial for studying invasion mechanisms driven by the surrounding microenvironment and for identifying biomarkers to diagnose and predict invasive cancer. In this study, we aimed to elucidate the regulation of cancer characteristics through the interactions between factors at the invasive tumor front and the surrounding tumor microenvironment. MATERIALS AND METHODS: The invasive tumor front (ITF) and tumor center (TC) of collective cancer invasion were analyzed using microarray to compare gene expression. A stable cell line with depleted DEFA1 expression was established, and its effect on cancer growth was observed using a mouse tongue xenograft model. Invasive activity was assessed using Transwell assays. Gene profiling of cancer cells and analysis of secreted proteins interacting with U937 monocytic cells during co-culture were conducted using QuantSeq 3' mRNA sequencing and LC-MS/MS analysis. RESULTS: DEFA1 was overexpressed at the ITF of collective cancer invasion. YD10B cells with depleted DEFA1 expression exhibited significantly reduced invasiveness and tumor growth without changes in the cell cycle distribution. Co-culture with U937 cells significantly enhanced the invasiveness of YD10B cells, which was inhibited by anti-DEFA1 treatment. QuantSeq 3' mRNA sequencing and LC-MS/MS analyses confirmed that DEFA1 derived from U937 cells increased the invasiveness of YD10B cells. Recombinant DEFA1 (rDEFA1) significantly enhanced the invasiveness of YD10B cells via the JNK MAPK/NF-[Formula: see text]B signaling pathway, independent of changes in DEFA1 expression within YD10B cells. CONCLUSION: DEFA1 is crucial for cancer invasion and growth, and monocyte-derived DEFA1 exacerbates these traits. This study highlights DEFA1's role in promoting invasion at the tumor front, where interactions with the microenvironment are active.