In this work, rice ultrafiltration albumin (RUA) and rice cadmium-binding protein (RCBP) were separated and purified from rice albumin (RA) in rice grain endosperm using ultrafiltration and ion-exchange chromatography (IEC). Physicochemical properties of RCBP were investigated, including cadmium content, molecular mass, ultraviolet absorption (UV) characteristics, amino acid composition, and secondary structure. The results showed that a novel cadmium-binding protein with the UV absorption peaks of 225 nm and 275 nm was purified from rice grain endosperm, and its molecular mass was determined as 13514 Da. RCBP had the highest glutamic acid content (18.31 g/100 g protein), followed by cysteine (11.45 g/100 g protein), as well as aromatic amino acids (tyrosine and phenylalanine) and histidine contents were 2.35 g/100 g protein and 1.76 g/100 g protein, respectively. In addition, the highest α-helix content (39.49%) was presented among secondary structures of RCBP, followed by random coiling (23.48%), and β-sheet and β-turn was 19.54% and 17.58%, respectively. Compared with RUA, cadmium (2.38 μg g−1) and cysteine contents of RCBP were increased by 2.66 and 5.26 times, whereas aromatic amino acids and lysine contents decreased by 46.10% and 40.97%, respectively. Moreover, the contents of α-helix and β-sheet increased significantly. Take together these results suggested that cadmium in RCBP may bind to cysteine and RCBP could be an atypical cadmium metallothionein (Cd-MT) in rice grain endosperm. Image 1073357 • A novel rice cadmium-binding protein (RCBP) was isolated and identified from rice grain endosperm. • The RCBP has 2 absorption peaks at 225 nm and 275 nm, respectively. • The RCBP has a low molecular mass with 13514 Da. • The structure of RCBP is more stable than rice ultrafiltration albumin (RUA) and rice albumin (RA). • The RCBP was regarded as an atypical cadmium-metallothionein. [ABSTRACT FROM AUTHOR]