C-terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine-phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk-interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk-binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk-SH2 domain-binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C-terminus was proved to directly bind to Csk-SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co-localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation-dependent manner and overexpression of Csk, but not its SH2-domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin-Darby canine kidney cells, implying the involvement of Csk-SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.