Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.