ABSTRACT: To determine the nanostructure of gelatin from catfish (Ictalurus punctatus) skin, atomic force microscopy (AFM) was used to study gelatin aggregates. The gelatin was extracted at an optimized acid concentration after alkaline processing. First, the AFM imaging parameters were optimized to obtain high-quality images. Then height mode with a 2-dimensional plane, 3-dimensional topographical images, and error signal mode images, which removed slow variations in surface topography but highlighted the edges of sample features, were used to analyze the structure of particles. The results describe fish gelatin at a nanoscale level for the first time and are compared with AFM images of mammalian gelatins. Both annular pores with diameters averaging 118 nm and spherical aggregates with an average diameter of 267 nm were seen in the AFM images of fish gelatin. From the AFM images, we propose that the structures formed were determined by whether the solution penetrated into the gelatin molecules evenly or not during hydrolysis. A scheme for the formation of annular pores and spherical aggregates is proposed.