Significance Most of the single miRNA gene knockouts display no developmental phenotype. Here, we report that simultaneous inactivation of two functionally overlapping miRNAs, miR-34b/c and miR-449, led to a sexually dimorphic partial perinatal lethality, growth retardation and sterility. Multiple underlying developmental defects, including underdevelopment of the basal forebrain structures, a lack of motile cilia in trachea and oviduct, severely disrupted spermatogenesis and oligoasthenoteratozoospermia, result from the dysregulation of ∼240 target genes that are mainly involved in three major cellular functions, including cell fate control, brain development and microtubule dynamics. This study provides physiological evidence demonstrating an essential role of miR-34b/c and miR-449 in normal brain development, motile ciliogenesis and spermatogenesis.