It is well established that the inclusion of amino acids in preimplantation embryo culture media can improve development rate and viability. Our recent studies have shown that embryos cultured in the amino acid L-proline from the 1-cell stage to the blastocyst stage, develop significantly better than embryos cultured with other amino acids. This study aimed to identify the pre-implantation stage(s) at which L-proline transport is required for development to be improved and then to identify and characterise the amino acid transporter responsible for L-proline uptake into the embryo. Mouse embryos were cultured in medium containing 400 µM L-proline either from the 1 cell to late 2-cell stage, or from the late 2-cell to blastocyst stage and then scored for development. Embryos that were cultured with L-proline in the later pre-implantation stages developed significantly better than embryos that were cultured with L-proline in the earlier stages, suggesting that the later pre-implantation stages are crucial for L-proline’s transport and subsequent effect. Competitive substrates were used to identify L-proline transporters in early mouse embryos. The presence of excess L-leucine or glycine in the culture medium prevented the improvement in development induced by L-proline. These findings implicated the transporters SLC6A19 (B⁰AT1) and SLC38A2 (SNAT2) since they transport L-proline, L-leucine and glycine. Immunostaining showed that B⁰AT1 was present in the pre-implantation embryo throughout all developmental stages and that it appeared to be expressed in the membrane from the 8-cell stage onwards. These results suggest that SLC6A19 (B⁰AT1) may be the transporter responsible for L-proline uptake in the pre-implantation embryo and for the subsequent improvement in embryo development.