The RNA binding protein heterogeneous nuclear ribonucleoprotein L-Like (hnRNP LL) is known to regulate the alternative splicing of various physiologically important precursor messenger RNAs (pre-mRNAs). It undergoes a wide range of post-translational modifications (PTMs), including phosphorylation, ubiquitination and acetylation. However, the target amino acids and effects of these PTMs on the functions of hnRNP LL have not been characterized so far. In this study, we show for the first time that the endogenous hnRNP LL is phosphorylated upon depolarization. Using phosphopeptide mapping followed by the generation of a custom-made phospho-site specific antibody, we further show that phosphorylation at Ser308 of hnRNP LL is induced by depolarization though it is probably not the major phospho-amino acid target of depolarization/CaMKIV. The residue is critical for the nuclear localization and its phosphorylation essential for the CaMKIV-caused perinucleolar localization of the hnRNP LL protein in HEK293T cells. The residue is likely also critical in the regulation of nuclear functions like pre-mRNA splicing.