Calreticulin (CRT) is the second most abundant protein within the endoplasmic reticulum (ER). Thanks to its ability to recognize monoglucosylated N-glycans, CRT plays a central role in the folding quality control of glycoproteins. Besides, due to its capacity to bind high amounts of calcium with low affinity, CRT is the principal calcium buffer of the ER. It was originally proposed that the lectin activity of CRT depends on calcium binding. By using a combination of biophysical techniques we found that both activities of CRT are mutually independent. In addition, we found that the calcium buffering domain of CRT allows its retrotranslocation into the cytosol in a process regulated by calcium. This process may be relevant for the crosstalk of different signal transduction pathways. Finally, these studies allowed us to develop a new sensor to measure macromolecular crowding in complex environments.