Non-small cell lung cancer (NSCLC) is the major cause of cancer death worldwide. Increasing evidence shows that microRNAs (miRNAs), evolutionally conserved non-coding RNAs, are widely involved in the development and progression of NSCLC. Aberrant alteration of miRNAs expression has been implicated in NSCLC initiation and progression. Herein, we studied the role of miR-27b in NSCLC cells. We found that miR-27b was significantly decreased in several NSCLC cell lines. Forced overexpression of miR-27 inhibited both the growth and invasion of NSCLC cells. Furthermore, we identified Sp1 transcription factor (Sp1) as a target of miR-27b in NSCLC cells. Moreover, we found that miR-27 suppressed growth and invasion of NSCLC cells partially by targeting Sp1. Our data indicate that miR-27b may play a critical role in the development of NSCLC.