To investigate the underlying molecular mechanism for connexin 26 (Cx26) knockout-induced apoptosis, we performed TUNEL assays to detect apoptosis in the cochlear sensory epithelium in Cx26 knockout mice. We also compared the miRNA expression profiles of Cx26 knockout and wild-type mice using microarray technology and bioinformatic analyses. Real-time PCR, luciferase reporter gene assays, and scala media microinjections were performed to identify the effect of a specific miRNA and its targets. The results showed that apoptosis increased in the cochlear sensory epithelium of Cx26 knockout mice. The abnormal expression of mir-27a and sgk1 in Cx26 knockout mice was verified with real-time PCR. Luciferase reporter gene assays showed that overexpression of mir-27a significantly decreased sgk1 reporter gene activity; an inhibitor of mir-27a blocked the effect. Mir-27a lentivirus also inhibited sgk1 expression in cultured cochlear tissue. Mir-27a shRNA treatment inhibited Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice and increased the expression of sgk1 mRNA. Thus, mir-27a was identified as an apoptotic molecule that participates in Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice by downregulating sgk1 expression.