In this study, we demonstrate that L-glutaminase, a marine bacterial enzyme with a molecular weight of 37 kDa, inhibits cancer cell proliferation in vitro through glutamine deprivation. The concentration of the enzyme reducing the viability of HeLa cells to 50% was determined to be 12.5 μg/mL; the function of L-glutaminase in controlling cell proliferation was further analysed by BrdU assays. To increase its stability and bioavailability, the enzyme was immobilized on polyethyleneglycol (PEG)-polyhydroxybutyrate (PHB) nanoparticles. A dented anatomy of the HeLa cells was observed under fluorescence and confocal microscopy when they were incubated with L-glutaminase and in glutamine-free medium, as also a 3-fold increase in caspase-3 activity was observed under the same conditions. Blebbed cytoplasm and shrunken nuclei were observed in treated cells under transmission electron microscopy (TEM). Finally, the influence of the enzyme on cell cycle and DNA damage was evaluated using flow cytometry and DNA fragmentation assays. The results confirmed significant damage to the DNA of HeLa cells incubated with L-glutaminase and in glutamine-free medium. These studies attest to the significant role played by L-glutaminase against proliferation in cancer cells through glutamine deprivation.