•Counter-current chromatography (CCC) was used to collect ganglioside fractions.•The selected CCC solvent system was 2:4:3:8 butanol/MTBE/acetonitrile/H2O.•0.5% (v/v) acetic acid was added to the lower phase in dual-mode elution CCC.•CCC separated mg levels of (d36:1) & (d38:1) homologues of porcine GM1, GD1a & GD1b.•The purity of ganglioside fractions was estimated using LC-MS/MS. Gangliosides play critical roles in the development of many progressive diseases. Due to their structural diversity, efficient methods are needed to separate individual gangliosides for studies of their functions, and for use as standards in the analysis of ganglioside mixtures. This proof-of-concept study reports a useful analytical-semi-preparative scale counter-current chromatography (CCC) enrichment of multiple ganglioside homologues of various species and classes at the milligram level. Since few individual ganglioside standards were available, this research aimed to achieve analytical-semi-preparative scale separation of gangliosides by differences in saccharide monomer compositions (classes), their arrangements (species), or ceramide compositions (homologues), using CCC. The solvent system composition, addition of solvent modifiers, and elution modes were all adjusted to separate porcine gangliosides, mainly GM1 (d36:1), GD1a (d36:1), GD1b (d36:1) and their (d38:1) homologues as a demonstration. The eluted compounds were analyzed by flow-injection analysis (FIA)-MS and LC-MS/MS. A two-phase solvent system, consisting of butanol/methyl t-butyl ether/acetonitrile/water at a ratio of 2:4:3:8 (v/v/v/v) with 0.5% (v/v) acetic acid added to the lower phase, was used to separate mg-levels of porcine gangliosides under dual-mode elution. The relative abundances of the above 6 gangliosides increased from 10 to 21% in the ganglioside extract to 55–73% in the collected fractions through the purification.