The placenta is important for pregnancy maintenance, and autophagy is documented to be essential for placental development. Autophagy is responsible for degrading and recycling cellular misfolded proteins and damaged organelles. Mitophagy is a selective type of autophagy, where the autophagic machinery engulfs the damaged mitochondria for degradation, and there is reciprocal crosstalk between autophagy and mitochondria. Within these processes, 5'-AMP-activated protein kinase (AMPK) plays an important role. However, the role of AMPK regulation in both autophagy and mitochondria in primary human trophoblasts is unknown. In this study, we address this question by investigating changes in mRNA expression and the abundance of autophagy- and mitochondria-related proteins in isolated human trophoblasts after treatment with AMPK agonists and antagonists. We found that compared to the control group, autophagy was slightly suppressed in the AMPK agonist group and significantly enhanced autophagy in the AMPK antagonist group. However, the expressions of genes related to autophagosome-lysosome fusion were reduced, while genes related to lysosomal function were unchanged in both groups. Furthermore, mitophagy and mitochondrial fusion/fission were both impaired in the AMPK agonist and antagonist groups. Although mitochondrial biogenesis was enhanced in both groups, the function of mitochondrial fatty acid oxidation was increased in the AMPK agonist group but decreased in the AMPK antagonist group. Overall, our study demonstrates that AMPK regulation negatively modulates autophagy and consequently affects mitophagy, mitochondrial fusion/fission, and function in primary human trophoblasts.