N-Alkylated 1,4-dideoxy-1,4-imino-L-lyxitols (N-alkylated polyhydroxypyrrolidines) differing in the alkyl chain length (from C7 to C14) and their capping functional group (methyl, amine, amidine and guanidine) have been synthesized. Based on molecular modeling, the structures were designed as selective inhibitors of Golgi α-mannosidase II (GMIIb) with a predicted reduced binding affinity toward lysosomal α-mannosidase II (LManII). In general, the potency of the derivatives toward GMIIb decreased in the following order: amidines > guanidines > alkanes > amines. The most potent inhibitors of GMIIb were N-nonylamidine and N-decylamidine 1,4-dideoxy-1,4-imino-L-lyxitols [K_i (GMIIb) = 4.0 μM and 5.5 μM]. Elongation of the alkyl chain led to an increase in potency while maintaining the selectivity toward the target enzyme. Molecular docking indicates that the alkyl chain length is crucial for an efficient interaction of the terminal group with a polar dyad (Asp270–Asp340) in the case of amidine, guanidine and amine structures and with a hydrophobic pocket (Pro298–Trp299) in the case of alkyl derivatives. Quantum mechanics calculations on conformational analysis of selected inhibitors indicate the differences in preferred envelope conformations for neutral and protonated forms of the pyrrolidine ring in a polar solution. NMR measurements in the polar solution at different pH values revealed that the N-substituted pyrrolidines with the terminal basic group may actually exhibit a different protonation form of the pyrrolidine ring under pH conditions occurring in Golgi apparatus compared with swainsonine or other N-substituted pyrrolidines lacking the terminal basic group.