KRIT1 plays a key role in regulating the barrier function of endothelial cells, where it localizes to the adherens junctions, cytoplasm, and nucleus. However, how subcellular localization may regulate KRIT1 remains unclear. Here, we investigate what effect nuclear localization has on its ability to stabilize the endothelial barrier. We generated a KRIT1 mutant lacking the endogenous nuclear localization signal (NLS) and a nuclear‐targeted KRIT1 isoform created by attaching the NLS of simian virus 40 and expressed these constructs in cells depleted of endogenous KRIT1. After confirming the relative non‐nuclear (KRIT1 ΔNLS ) and nuclear (KRIT1 NLS+ ) enrichment of these constructs using confocal microscopy and cellular fractionation, we assessed whether these constructs were able to functionally rescue phenotypes characteristic of KRIT1‐depleted cells. Our results showed that nuclear localized KRIT1 remains fully functional. However, the KRIT1 ΔNLS construct failed to rescue the KRIT1 depletion phenotype. KRIT1 ΔNLS also disrupted binding to ICAP1α, as shown by co‐immunoprecipitation. To determine whether the loss of function was due to loss of ICAP1α‐mediated conformational change or altered localization, we incorporated two mutants known to disrupt the N‐ to C‐terminal interaction in KRIT1, 192 NPXY 195 → APAA or W688A into our KRIT1 ΔNLS and KRIT1 NLS+ constructs. The presence of these additional mutations had unexpected effects on nuclear localization of KRIT1 ΔNLS and KRIT1 NLS+ but were able to restore barrier‐stabilizing function to KRIT1 ΔNLS . Overall, our findings provide key insights into the role of ICAP1α binding and nuclear localization in the regulation of KRIT1 and raise new questions regarding potentially novel functions of KRIT1 in the nucleus.