Nowadays, probiotics has been proposed for substituting antibiotics in animal feed, since the European Union (EU) banned the latter compounds in 2006 to avoid their serious side effects on human health. Therefore, this work aimed to produce a probiotic product by fed-fermentation of whey with kefir grains for use in animal feed. The whey was fermented with a combination of kefir grains AGK1 and the free biomass present in whole milk fermented milk used to activate these kefir grains. The probiotic culture obtained was characterized with high levels of biomass, total viability and antibacterial activity. Some probiotic properties of the probiotic culture were investigated in vitro, including its survival at low pH values, under simulated gastrointestinal conditions, after freezing in skim milk at − 20 ºC, and in the commercial feed during storage at room temperature. The viable cells of lactic and acetic acid bacteria and yeasts exhibited higher tolerance to acidic pH and simulated gastrointestinal conditions when the cells were protected with skim milk and piglet feed, compared with washed cells. The results indicated the feasibility of producing a probiotic product at a low cost with a potential application in animal feed.