<p>Peroxisomes are small, highly dynamic organelles involved in a plethora of metabolic pathways. They are essential for the efficient exchange of metabolites and cellular messengers orchestrating intracellular signaling. Calcium (Ca²⁺) is one of the most prominent physiological signaling elements and regulates a wide variety of processes in cellular homeostasis and function. Recently, we showed that peroxisomes participate in cellular Ca²⁺ dynamics by taking up and releasing Ca²⁺ following store-operated calcium entry (SOCE), however, the mechanism of peroxisomal Ca²⁺ uptake and its modulators remained unknown. Using live cell imaging in combination with genetically encoded calcium indicators (GECI), we show that peroxisomal calcium dynamics are independent of PEX11β and the pore protein PXMP2. Instead, we find that the ACBD5-dependent membrane contact site between peroxisomes and the endoplasmic reticulum (ER) is necessary for efficient peroxisomal Ca²⁺ uptake. Further, we identify the ACBD5-dependent peroxisome-ER contact site as the major factor restricting peroxisome motility within the cell. Microtubules and SOCE stimulation exert smaller and independent effects on peroxisome motility. This work expands the range of known functions of the peroxisome-ER contact site.</p>