Cadherin-17 (CDH17) is highly overexpressed in gastrointestinal malignancies such as colorectal, gastric, and pancreatic cancer. CDH17 is a promising next-generation cancer target, offering high tumor selectivity, low normal-tissue expression, and broad compatibility with antibody, ADC, and bispecific platforms. Based on this rationale, we developed a CDH17-targeted antibody, TLT-001, which demonstrated high-affinity, stable binding to CDH17 (KD = 5.85 × 10⁻¹² M) and selective reactivity toward CDH17-positive gastric and pancreatic cancer cell lines (AGS, AsPC-1) relative to CDH17-negative cells, along with robust, time-dependent receptor-mediated internalization in CDH17-expressing lines and negligible uptake in CDH17-low/negative lines. An MMAF-conjugated ADC generated from TLT-001 induced potent, dose-dependent cytotoxicity in CDH17-positive cells (IC₅₀ = 160 pM), whereas the naked antibody and free payload alone showed minimal effect, and this cytotoxic activity was significantly attenuated upon CDH17 knockdown, confirming a target-specific mechanism of action. Collectively, these findings establish CDH17 as a structurally and functionally distinct, cancer-selective therapeutic target and demonstrate that the CDH17-targeted ADC TLT-001 exerts potent, receptor-mediated, target-dependent cytotoxicity, supporting its continued preclinical development as a novel ADC therapeutic for CDH17-overexpressing gastrointestinal cancers.