Nostoc commune has been traditionally used in China as a health food and medicine. The water stress proteins (WSP) of Nostoc commune are the major component of the extracellular matrix. This study purified and identified the water stress proteins (WSP1) from Nostoc commune Vauch., which could inhibit the proliferation of human colon cancer cell lines. The IC50 values of WSP1 against DLD1, HCT116, HT29, and SW480 cells were 0.19 ± 0.02, 0.21 ± 0.03, 0.39 ± 0.05, and 0.41 ± 0.01 μg/μL, respectively. Notably, it displayed very little effect on the normal human intestinal epithelial FHC cell line. The IC50 value of WSP1 against FHC cells was 0.67 ± 0.05 μg/μL. Moreover, the growth of DLD1 xenografted tumors in nude mice were significantly suppressed in the WSP1 treated group. Mechanistically, the cell-cycle analysis revealed that WSP1 induced growth inhibition by G1/S arrest. Meanwhile, Western blotting and immunohistochemistry assays showed WSP1 could activate caspase-8, -9, and -3, along with subsequent PARP cleavage. Furthermore, the pan-caspase inhibitor, z-VAD-FMK, partly reversed the effect caused by WSP1, confirming that WSP1 induced cell apoptosis through caspase-dependent pathway. Collectively, WSP1 has targeted inhibition for colon cancer proliferation both in vitro and in vivo and it is valuable for future exploitation and utilization as an antitumor agent.