Previous studies from our laboratory have demonstrated that the coupling of doxorubicin with SynB1 vector dramatically increases its brain uptake. In the present study, we have evaluated the broad application of this approach using another molecule: benzylpenicillin (B-Pc). We, therefore, have coupled the beta-lactam antibiotic B-Pc with SynB1 and assessed its ability to cross the blood-brain barrier (BBB) using the in situ rat brain perfusion method. We first confirmed the very low brain uptake of free radiolabeled B-Pc. When B-Pc was coupled to SynB1, its uptake in brain was increased by a factor of 7, without compromising the BBB integrity. The vectorised B-Pc was distributed in all the gray areas assessed (frontal, parietal, and occipital cortex, thalamus, hippocampus, and striatum). Moreover, using a wash-out procedure and a capillary depletion method, we have shown that the radiolabeled B-Pc was associated mainly with brain parenchyma. In summary, this study demonstrates the successful application of the use of SynB1 vector for the transport of B-Pc across the BBB.