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Background: Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells., Methods: Stable isotope metabolic labeling, media creatine depletion, and Na + /K + -ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na + /K + -ATPase and creatine uptake., Results: Inhibition of the Na + /K + -ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na + /creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na + /K + -ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na + /K + -ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na + /K + -ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells., Conclusions: These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na + /K + -ATPase pump activity., General Significance: These studies identify kinase regulation of the Na + /K + -ATPase as pivotal in regulating creatine uptake and energy metabolism in cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)