Xq28 (involving MECP2) duplication syndrome is a severe neurodevelopmental disorder in males, most females are asymptomatic carriers, but there are phenotypic heterogeneities in the females. Skewed X-chromosome inactivation (XCI) seems to prevent duplicated region activation in asymptomatic females, but it remains controversial. Herein we reported two asymptomatic females (daughter and mother) with interstitial Xq28 duplication. HUMARA and RP2 assays showed that both had complete skewed XCI, the Xq28 duplicated chromosome was inactivated in the daughter, but surprisingly, it was activated in her mother. Interestingly, by combining RNA sequencing and whole-exome sequencing, we confirmed that XIST only expressed in the Xq28 duplication chromosomes of the two females, indicating that the Xq28 duplication chromosomes were inactive. Meanwhile, MECP2 and most XCI genes in the duplicated X-chromosomes were not transcriptionally expressed or upregulated, precluding major clinical phenotypes in the two females, especially the mother. We showed that XCI status detected by RNA sequencing was more relevant for establishing the clinical phenotype of MECP2 duplication females. It suggested there were other factors maintaining the XCI status in addition to DNA methylation, a possible additional inhibition mechanism occured at the transcriptional level in the unmethylated X-chromosome, counter balancing the MECP2 duplication’s detrimental phenotype effects