The aim of this study was to detect mutations of the nuclear factor I/X (Nfix) gene and examine the association of its polymorphisms with growth traits in cattle. Six sequence variants (SVs) including five single-nucleotide mutations and an indel with multiple alleles were detected, among which four polymorphisms within the Nfix gene were identified in 1159 individuals of five cattle breeds by sequencing and forced PCR-RFLP methods. The results of haplotype analysis showed 14 haplotypes within the breeds. Three haplotypes were shared by the five cattle breeds. Hap1 (ACAI) was extremely predominant in all test populations, which suggested that individuals with Hap1 (ACAI) were more adapted to the steppe environment. Association analysis in Nanyang cattle showed that two SVs of the Nfix gene were significantly associated with growth traits at different ages. In addition, the locations of the SVs showed that the 3' terminal of the bovine Nfix gene was unstable. Combining this instability with its characteristic of multiple alternative splicing, we conjectured that some SVs might have a relationship with the formation of the splices through which growth traits are modulated. This study will provide useful information for the selection and detection of multiple forms of alternative splicing of the bovine Nfix gene.