--- - |2+ Key points Amino acid absorption requires luminal uptake into and subsequent basolateral efflux out of epithelial cells, with the latter step being critical to regulate the intracellular concentration of the amino acids. The basolateral essential neutral amino acid uniporter LAT4 (SLC43A2) has been suggested to drive the net efflux of non‐essential and cationic amino acids via parallel amino acid antiporters by recycling some of their substrates; its deletion has been shown to cause defective postnatal growth and death in mice. Here we test the regulatory function of LAT4 phosphorylation sites by mimicking their phosphorylated and dephosphorylated states in Xenopus laevis oocytes and show that dephosphorylation of S274 and phosphorylation of S297 increase LAT4 membrane localization and function. Using new phosphorylation site‐specific antibodies, we observe changes in LAT4 phosphorylation in mouse small intestine that correspond to its upregulation at the expected feeding time. These results strongly suggest that LAT4 phosphorylation participates in the regulation of transepithelial amino acid absorption. Abstract The essential amino acid uniporters LAT4 and TAT1 are located at the basolateral side of intestinal and kidney epithelial cells and their transport function has been suggested to control the transepithelial (re)absorption of neutral and possibly also cationic amino acids. Uniporter LAT4 selectively transports the branched chain amino acids leucine, isoleucine and valine, and additionally methionine and phenylalanine. Its deletion leads to a postnatal growth failure and early death in mice. Since LAT4 has been reported to be phosphorylated in vivo, we hypothesized that phosphorylation regulates its function. Using Xenopus laevis oocytes, we tested the impact of LAT4 phosphorylation at Ser274 and Ser297 by expressing mutant constructs mimicking phosphorylated and dephosphorylated states. We then investigated the in vivo regulation of LAT4 in mouse small intestine using new phosphorylation site‐specific antibodies and a time‐restricted diet. In Xenopus oocytes, mimicking non‐phosphorylation of Ser274 led to an increase in affinity and apparent surface membrane localization of LAT4, stimulating its transport activity, while the same mutation of Ser297 decreased LAT4's apparent surface expression and transport rate. In wild‐type mice, LAT4 phosphorylation on Ser274 was uniform at the beginning of the inactive phase (ZT0). In contrast, at the beginning of the active phase (ZT12), corresponding to the anticipated feeding time, Ser274 phosphorylation was decreased and restricted to relatively large patches of cells, while Ser297 phosphorylation was increased. We conclude that phosphorylation of small intestinal LAT4 is under food‐entrained circadian control, leading presumably to an upregulation of LAT4 function at the anticipated feeding time. - 'The Journal of Physiology, EarlyView. '