Introduction The processing of annatto seeds yields a low-cost by-product known as annatto seed by-product. Due to its remarkable bixin levels, which is a natural antioxidant, the annatto seed by-product could be used in animal feed supplemen- tation. As a low-moisture product, it is important to know information about hygroscopic behavior, changes during storage, package selection and drying equip- ment design. Objectives The annatto seed by-product's hygroscopic behavior was studied at 25 °C and 35 °C by using saturated salt solutions and the static method. Methods During storage with different relative air humidity, the annatto seed by-product's isosteric heat of sorption, bixin content and instrumental color parameters were evaluated. Results The isotherms exhibited type III behavior and the Kuhn model properly fitted the annatto seed by-product's equilibrium mois- ture data. Above 75% air relative humidity, agglomeration, darkening and fungal development were observed. The isosteric heat of sorption decreased with an increase in moisture content. Conclusion Once the equilibrium was established, bixin losses (approximately 43% of the content) and color changes were more prominent at 35 °C, especially under 68% relative humidity.