Alport syndrome (AS) is an inherited disease of collagen type IV causing progressive renal disease, sensorineural hearing loss, and ocular abnormalities with variable severity. Here we report a COL4A5 aberrant splicing mutation leading to X-linked Alport syndrome (XLAS) in two children of an Iranian non-consanguineous couple. The proband was a 10-year-old boy with clinical manifestations of hematuria, proteinuria, and mild to moderate bilateral sensorineural hearing loss (BSNHL), who was diagnosed with AS based on renal pathology and genetic testing. Whole exome sequencing of the proband identified a hemizygous canonical splice site variant; c.4994+1G>A, in intron 52 of the COL4A5 gene. The same mutation was detected in his carrier mother and affected brother by Sanger sequencing. Thus, confirming the diagnosis of XLAS in the two individuals with renal impairment in this family. Our results extend the range of COL4A5 splicing mutations and highlight their importance.