Hsp90 beta is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90 beta for proper folding and/or activity. Regulation of Hsp90 beta is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90 beta, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90 beta is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90 beta activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90 beta globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90 beta and its secretion, through changes in the conformation of the chaperone.