Lactobacilli have been shown to inhibit or suppress cancer cell growth through the release of strain-specific bioactive metabolites and their inclusion in functional foods could exert a health promoting activity on human health. Herein, we examined the antiproliferative activity of the Lactiplantibacillus plantarum strains S2T10D and O2T60C, which have been previously shown to exert different butyrogenic activities. Human HT-29 cells were employed as an in vitro colon cancer model and both bacterial strains were found to inhibit their growth. However, the strain S2T10D showed a greater antiproliferative activity which, interestingly, was correlated to its butyrogenic capability. Noteworthy, for the non-butyrogenic strain O2T60C, the growth inhibitory capability was rather limited. Furthermore, both the butyrate-containing supernatant of S2T10D and glucose-deprived cell culture medium supplemented with the same concentration of butyrate found in S2T10D supernatant, induced a pH-independent cancer cell growth inhibition accompanied by downregulation of cyclin D1 at mRNA level. The downregulation of cyclin D1 gene expression was accompanied by cell cycle arrest in G2/M phase and decrease of cyclin B1 and D1 protein levels. This in vitro study underlines the impact of Lpb. plantarum in the growth inhibition of cancer cells, and proposes butyrate-mediated cell cycle regulation as a potential involved mechanism. Since the production of butyric acid in Lpb. plantarum has been proven strain-dependent and differentially boosted by specific prebiotic compounds, our results open future research paths to determine whether this metabolic activity could be modulated in vivo by enhancing this antiproliferative effects on cancer cells.