Two estrogen receptors, ESR1 and ESR2, are responsible for the classical actions of estrogens in mammalian species. They display different spatiotemporal expression patterns and nonoverlapping functions in various tissues and physiological conditions. In this study, a novel knock‐in mouse line that expresses codon‐improved Cre recombinase (iCre) under regulation of the natural Esr1 promoter (Esr1–iCre) was developed. Functional characterization of iCre expression by crossing them with reporter lines (ROSA26‐lacZ or Ai9‐RFP) showed that iCre is faithfully expressed in Esr1‐lineage cells. This novel transgenic mouse line will be a useful animal model for lineage‐tracing Esr1‐expressing cells, selective gene ablation in the Esr1‐lineage cells and for generating global Esr1 knockout mice.