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Objectives To investigate the bio-functions and the molecular mechanisms of NIN1/proteasome 26S subunit non-ATPase 8 binding protein 1 homolog (NOB1) in colorectal cancer cells. Methods NOB1 expression was silenced using si-RNA in SW480 and LoVo cells. The transfection efficiency was measured by western blotting and RT-qPCR. Subsequently, the proliferation of SW480 and LoVo cells was determined using both MTT assay and colony-formation assay. Apoptosis and cell cycle analysis were determined using flow cytometry. Results Compared with the normal control (NC) and scramble cells, si-NOB1 could significantly attenuate the proliferation, colony-formation ability and cell percentage of S stage (p < 0.05). Additionally, at the phosphorylation level, si-NOB1 could notably increase the expression of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. Conclusions Inhibition of NOB1 expression suppressed the proliferation, and promoted the apoptosis through regulation of the JNK signaling pathway.