Buttermilk, a co-product generated during butter production, has high nutritional value; however, it is often underutilized by small- and medium-sized dairy industries in developing countries. This study aimed to evaluate the use of different proportions of whole milk and buttermilk (100:0; 80:20; 60:40; 40:60; 20:80; and 0:100) for kefir production, with the goal of adding value to this co-product and promoting bioeconomy principles in the dairy sector. A 100% milk formulation was used as the control. The proximate composition, kefir grain biomass growth over 21 days, syneresis, and water retention capacity (WRC) were evaluated. The results showed that increasing the proportion of buttermilk led to a linear increase in moisture content (from 89.70% to 96.57%) and a significant decrease in fat (from 3.05% to 0.55%), protein (from 3.43% to 1.89%), and carbohydrate content (from 3.34% to 0.63%). Regarding kefir grain growth, formulations with higher milk content (100%, 80%, and 60%) showed the greatest increases in biomass (150.00 g, 105.50 g, and 107.60 g), respectively. Conversely, kefir produced exclusively with buttermilk (100%) reached only 16.62 g, indicating that whole milk provides more favorable nutritional conditions for grain development. WRC decreased with increasing amounts of buttermilk: 100% milk (52.42%), 80% (42.42%), 60% (33.91%), 40% (29.55%), 20% (23.01%), and 0% milk (18.58%). This is likely due to buttermilk's lower protein content (1.76%) compared to milk (3.00%). On the other hand, syneresis increased proportionally with the buttermilk content, ranging from 31.92% (100% milk) to 73.92% (100% buttermilk), suggesting weaker and less stable gel structures favoring greater phase separation. Therefore, partial replacement of milk by buttermilk in kefir production proved viable up to the 40% level, maintaining acceptable physicochemical and technological characteristics. The inclusion of buttermilk contributes to the valorization of an underutilized co-product, promoting sustainable alternatives aligned with bioeconomy principles in the dairy industry.