The biogenic polyamines, spermine (Spm) and spermidine, are organic polycations present in millimolar concentrations in all eukaryotic cells participating in the regulation of vital cellular functions including proliferation and differentiation. The design and biochemical evaluation of polyamine analogues are cornerstones of polyamine research. Here we synthesized and studied novel C-methylated Spm analogues: 2,11-dimethylspermine (2,11-Me<sub>2</sub>Spm), 3,10-dimethylspermine (3,10-Me<sub>2</sub>Spm), 2-methylspermine, and 2,2-dimethylspermine. The tested analogues overcame growth arrest induced by a 72 h treatment with α-difluoromethylornithine, an ornithine decarboxylase (ODC) inhibitor, and entered into DU145 cells via the polyamine transporter. 3,10-Me<sub>2</sub>Spm was a poor substrate of spermine oxidase and spermidine/spermine-<i>N</i><sup>1</sup>-acetyltransferase (SSAT) when compared with 2,11-Me<sub>2</sub>Spm, thus resembling 1,12-dimethylspermine, which lacks the substrate properties required for the SSAT reaction. The antizyme (OAZ1)-mediated downregulation of ODC and inhibition of polyamine transport are crucial in the maintenance of polyamine homeostasis. Interestingly, 3,10-Me<sub>2</sub>Spm was found to be the first Spm analogue that did not induce OAZ1 and, consequently, was a weak downregulator of ODC activity in DU145 cells.