GM3, a major lipid component of the plasma membrane outer leaflet in mammalian cells, is synthesized in the luminal side of Golgi by ST3GAL5 protein (ST3G5), a type II membrane protein. Two strains of St3Gal5 knockout mice have been established for studies of GM3 physiological function: St3Gal5-Ex5-KO (lacking exon 5, which contains the catalytic domain of ST3G5), and St3Gal5-Ex3-KO (lacking exon 3, which contains the initiation codons). Results of the present study demonstrate that GM3 synthesis is still present, at a low level, in liver of St3Gal5-Ex3-KO mice. St3Gal5 has two mRNA transcriptional variants: a-type and b-type. When exon 3 is deleted, ST3G5 is not translated from a-type or b-type, as a result of initiation codon deletion or frame shift. Through NCBI database search and real-time PCR analyses of various mouse tissues, we identified a liver-specific St3Gal5 transcriptional variant (c-type) capable of producing artificial ST3G5 (M*-ST3G5) having GM3 synthase activity in the absence of exon 3. St3Gal5-Ex3-KO mice expressed c-type mRNA without exon 3 (c-type−/−) in liver. The transmembrane and catalytic domains of M*-ST3G5 translated from c-type−/− were identical to those from wild-type, although the cytoplasmic regions differed. Expression of M*-ST3G5 in embryonic fibroblasts derived from St3Gal5-Ex3-KO mice led to GM3 synthesis; M*-ST3G5 thus displayed enzyme activity in vivo. Taken together, our findings indicate that expression of liver-specific c-type variant accounts for the residual GM3 synthase activity observed in liver of St3Gal5-Ex3-KO mice.