Cats with suspicion of feline leukemia virus (FeLV) infection are commonly tested for antigenemia by p27 ELISA. However, many p27 negative cats test FeLV provirus positive in the blood. So far, the risk of a FeLV transmission via provirus-positive blood has not been evaluated. The aim of this study was to explore this risk. Fifteen ten-week old specified pathogen free (SPF) kittens were randomly assigned to three groups: five cats (group A) received blood from a FeLV provirus positive cat, five cats (group B) were transfused with blood from a provirus and viral RNA positive cat and five control cats (group C) received SPF blood. All cats in groups A and B turned provirus and plasma viral RNA positive post transfusion. Moreover, all cats in group A, and three cats in group B became p27 positive. All cats in group C stayed FeLV negative. Remarkably, cats in group A turned FeLV positive later than cats in group B but had a graver infection outcome (higher FeLV loads); two cats in group A became persistently infected. Our results demonstrate for the first time that FeLV infection can be transmitted via blood transfusion of blood from FeLV provirus positive, antigen negative cats to naïve recipients. Based on our results we highly recommend screening blood donors for FeLV provirus by PCR prior to blood transfusion.