p16INK4a plays a key role in control of cell cycle progression by negatively regulating the CDK4/6 activity. This study establishes that the p16INK4a minimal promoter region required for the transcription factor Sp1 function is mapped at 62 bp upstream of the translation initiation codon. This region is GC-rich and shown to interact specifically with Sp1. siRNA-induced Sp1 silencing resulted in the inhibition of the p16INK4a minimal promoter activity. Additionally, by using a promoter sequence-directed siRNA method, we demonstrate that the histone H3 at the GC-rich region in the minimal promoter of p16INK4a is hypermethylated, with a concurrent reduction of both the activity of p16INK4a promoter and the level of endogenous p16INK4a mRNA. Moreover, we show that the specific mutation of the GC-rich region of the minimal promoter resulted in the complete loss of its regulatory activities. We conclude that the region spanning −62 to +1 bp of p16INK4a promoter plays a role in p16INK4a transcription regulation.