Eisosomes help sequester a subgroup of plasma membrane proteins into discrete membrane domains that colocalize with sites of endocytosis. Here we show that the major eisosome component Pil1 in vivo is a target of the long-chain base (LCB, the biosynthetic precursors to sphingolipids)-signaling pathway mediated by the Pkh-kinases. Eisosomes disassemble if Pil1 is hyperphosphorylated (i) upon overexpression of Pkh-kinases, (ii) upon reducing LCB concentrations by inhibiting serine-palmitoyl transferase in lcb1-mutant cells or by poisoning the enzyme with myriocin, and (iii) upon mimicking hyperphosphorylation in pil1-mutant cells. Conversely, more Pil1 assembles into eisosomes if Pil1 is hypophosphorylated (i) upon reducing Pkh-kinase activity in pkh1 pkh2-mutant cells, (ii) upon activating Pkh-kinases by addition of LCBs, and (iii) upon mimicking hypophosphorylation in pil1-mutant cells. The resulting enlarged eisosomes show altered organization. Other data suggest that Pkh signaling and sphingolipids are important for endocytosis. Taken together with our previous results that link eisosomes to endocytosis, these observations suggest that Pkh-kinase signaling relayed to Pil1 may help regulate endocytic events to modulate the organization of the plasma membrane.