During the peri-implantation period of pregnancy, the trophectoderm of pig conceptuses utilize glucose via multiple biosynthetic pathways to support elongation and implantation, resulting in limited availability of pyruvate for metabolism via the TCA cycle. Therefore, we hypothesized that porcine trophectoderm cells replenish tricarboxylic acid (TCA) cycle intermediates via a process known as anaplerosis and that trophectoderm cells convert glutamine to α-ketoglutarate, a TCA cycle intermediate, through glutaminolysis. Results demonstrate: (1) that expression of glutaminase (GLS) increases in trophectoderm and glutamine synthetase (GLUL) increases in extra-embryonic endoderm of conceptuses, suggesting that extra-embryonic endoderm synthesizes glutamine, and trophectoderm converts glutamine into glutamate; and (2) that expression of glutamate dehydrogenase 1 (GLUD1) decreases and expression of aminotransferases including PSAT1 increase in trophectoderm, suggesting that glutaminolysis occurs in the trophectoderm through the GLS-aminotransferase pathway during the peri-implantation period. We then incubated porcine conceptuses with 13C-glutamine in the presence or absence of glucose in the culture media and then monitored the movement of glutamine-derived carbons through metabolic intermediates within glutaminolysis and the TCA cycle. The 13C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of 13C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux.