Heterosis has been used widely in the breeding of maize and other crops and plays an important role in increasing yield, improving quality, and enhancing stress resistance, but its molecular mechanism is far from clear. To determine whether microRNA (miRNA)-dependent gene regulation is responsible for heterosis of elongating internodes below the ear and ear height in maize, a deep-sequencing strategy was applied to the elite hybrid Xundan20, which is currently cultivated widely in China, and its two parents. RNA was extracted from the eighth internode because it shows clear internode length heterosis. A total of 99 conserved maize miRNAs were detected in both the hybrid and parental lines. Most of these miRNAs were expressed nonadditively in the hybrid compared with its parental lines. These results indicated that miRNAs might participate in heterosis during internode expansion in maize and exert an influence on ear and plant height via the repression of their target genes. In total, eight novel miRNAs belonging to four miRNA families were predicted in the expanding internode. Global repression of miRNAs in the hybrid, which might result in enhanced gene expression, might be one reason why the hybrid shows longer internodes and taller seedlings compared with its parental lines.