Ubiquitin-specific protease (USP7) is an oncogene that is involved in many tumour types including colorectal cancer by dysregulating various cellular pathways such as canonical Wnt signalling and the P53-MDM2 pathway. Discovering novel USP7 interacting partners can reveal the implications for the role of USP7 in other pathways and new biological functions. Therefore, USP7 becomes a promising target for cancer therapy. In this work, knocked-down USP7 colorectal cancer cells (LS188) were used as a cell model and liquid chromatography mass spectrometry was performed. We found that USP7 knockdown significantly inhibited colorectal cancer cell viability, colony formation, cell cycle and cell adhesion. The effect of USP7 Knockdown and inhibition on cell viability has been studied in both two-dimensional (2D) and three-dimensional culture (3D). The impact on the cell cycle in USP7 inhibited or depleted cells was p53 independent. The novelty in our study is that we found that protein expression of Ajuba LIM domain protein was reduced upon USP7 depletion by shRNA or siRNA as well as inhibition with FT671 inhibitor. This reduction of Ajuba protein levels was confirmed using immunofluorescence confocal microscopy. Co-immunoprecipitation revealed that USP7 is associated with Ajuba in vitro while Knocking down Ajuba does not affect USP7 expression. Moreover, quantitative PCR analysis showed that USP7 does not affect Ajuba mRNA levels meaning that USP7 plays a role in the post-transcriptional of Ajuba. Our findings underline the functions and oncogenic roles of USP7 through its protein interaction networks and that Ajuba is one of the USP7 protein networks present in colorectal cancer.