This dataset was generated to identify phosphorylation sites of ZO-1 regulated by ERK signaling. Cells were treated with TPA alone or with TPA in combination with the MEK inhibitor PD0325901, followed by phosphopeptide enrichment and LC–MS/MS analysis. TPA activates multiple kinases including ERK, whereas PD0325901 selectively inhibits MEK, the upstream activator of ERK. Thus, ERK activity is specifically suppressed in the co-treated condition. Phosphorylation sites detected in the TPA-treated samples but not in the TPA + PD0325901-treated samples are considered to be associated with ERK-dependent phosphorylation of ZO-1.