Cytoplasmic FMR1-interacting protein family contains two members, CYFIP1 and CYFIP2, evolutionarily conserved multifunctional proteins. Despite their high sequence homology, varied evidence indicates that CYFIP1 and CYFIP2 have distinct functions in vivo and are linked to different brain disorders. However, the underlying mechanisms remain largely unknown.We conducted reciprocal immunoprecipitation experiments to explore the underlying mechanisms using CYFIP1-2×Myc and CYFIP2-3×Flag knock-in mice. The results revealed that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Subsequent mass spectrometry analysis of the interactomes of CYFIP2 and CYFIP2, using immunoprecipitation samples, demonstrated that the two proteins have different interactome partners. Investigations in single-cell RNA-sequencing databases suggested enriched expression of Cyfip1 in non-neuronal cells and Cyfip2 in neurons, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, indicating the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome and co-expression analysis of Cyfip1 with astrocytic genes consistently linked CYFIP1 with focal adhesion proteins. The partial co-localization between CYFIP1 and focal adhesion proteins was verified in vitro.Overall, these results imply a distinct role for CYFIP1 associated with astrocytic focal adhesion, potentially contributing to the diverse brain functions and dysfunctions of CYFIP1 and CYFIP2.