Background/Aims: Transport regulation involves several kinases including SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether SPAK and/or OSR1 participate in the regulation of the creatine transporter CreaT (SLC6A8), which accomplishes Na<sup>+</sup> coupled cellular uptake of creatine in several tissues including kidney, intestine, heart, skeletal muscle and brain. Methods: cRNA encoding SLC6A8 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active <sup>T233E</sup>SPAK, WNK insensitive <sup>T233A</sup>SPAK, catalytically inactive <sup>D212A</sup>SPAK, wild-type OSR1, constitutively active <sup>T185E</sup>OSR1, WNK insensitive <sup>T185A</sup>OSR1 and catalytically inactive <sup>D164A</sup>OSR1. Transporter activity was determined from creatine (1 mM) induced current utilizing dual electrode voltage clamp. Results: Coexpression of wild-type SPAK and of <sup>T233E</sup>SPAK, but not of <sup>T233A</sup>SPAK or of <sup>D212A</sup>SPAK was followed by a significant decrease of creatine induced current in SLC6A8 expressing oocytes. Coexpression of SPAK significantly decreased maximal transport rate. Coexpression of wild-type OSR1, <sup>T185E</sup>OSR1 and <sup>T185A</sup>OSR1 but not of <sup>D164A</sup>OSR1 significantly negatively regulated SLC6A8 activity. OSR1 again decreased significantly maximal transport rate. Conclusions: Both, SPAK and OSR1, are negative regulators of the creatine transporter SLC6A8.