Tagging of individual proteins with genetically encoded fluorescent proteins (FPs) has been used extensively to study localization and interactions in live cells. Recent developments in single-molecule localization microscopy have enabled the dynamic visualization of individual tagged proteins inside living cells. However, tagging proteins with FPs is not without problems: formation of insoluble aggregates and inhibition of native functions of the protein are well-known issues. Previously reported artifacts manifest themselves at all expression levels of the FP-tagged proteins, making the design of control experiments relatively straightforward. Here, we describe a previously uncharacterized mislocalization artifact of Entacmaea quadricolor red fluorescent protein variants that is detectable at the single-molecule level in live Escherichia coli cells.