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The GPAT4 gene is considered as a potential functional candidate for single nucleotide polymorphism (SNP) studies in dairy cattle breeding due to its association with dairy performance in cattle by encoding an enzyme responsible for the presence of diacylglycerols and triacylglycerols in milk. Using the example of the GPAT4 gene, we applied the minigene splicing assay to analyze the functional consequences of its variant that was predicted to affect normal splicing. The results of functional analysis revealed the sequence variations (rs442541537), transfection experiments in a wild type and mutant cell line model system demonstrated that the investigated mutation in the second intron of the GPAT4 gene was responsible for the presence of a second exon in mature messenger RNA (mRNA). The cases of its absence in the spliced mature mRNA transcript resulted in a truncated dysfunctional protein due to the appearance of a stop codon. Thus, the discovered SNP led to alternative splicing in pre-mRNA by the 'cassette exon' ('exon skipping') mechanism. The studied mutation can potentially be a molecular genetic marker for alternative splicing for the GPAT4 gene and, therefore contributes to economic benefits in cattle breeding programs.