This study investigates the ability of a near-infrared fluorescence (NIRF) imaging agent, ASP5354, for in vivo fluorescence imaging of esophageal squamous cell carcinoma (ESCC) tissues. The ability of ASP5354 was evaluated using a single dose of ASP5354 or indocyanine green (ICG), which was intravenously administered to a KYSE850 human ESCC xenograft mouse model. Subsequently, in vivo NIRF images of the mouse were obtained using a clinically available camera system. ASP5354-specific NIRF signals were strongly detectable in KYSE850 carcinoma tissues immediately (30 s) following ASP5354 administration compared with normal tissues. Meanwhile, ICG could not distinguish between normal and carcinomatous tissues. To elucidate the associated imaging mechanisms, the vascular permeability of ASP5354 and ICG was investigated in rat back dermis treated with saline or histamine, which enhances vascular permeability, using in vivo NIRF imaging. ASP5354 exhibited higher vascular permeability in histamine-treated skin than in normal skin. KYSE850 carcinoma tissues can be distinguished from normal tissues based on the measurement of ASP5354-specific NIRF signals, and the mechanism that enables imaging relies on the specific and rapid leakage of ASP5354 from the capillaries into the stroma of carcinoma tissues.