High temperature requirement factor A4 (HtrA4) is a member of the HtrA family of serine peptidases involved in regulating protein‑protein interactions. Little is known regarding the function of HtrA4 in humans and in mouse models. To gain insights into the role of HtrA4 in vivo, mice were generated with a conditional null allele of HtrA4 by flanking exons 4, 5 and 6 with loxP sites. Cre‑mediated recombination, using a ubiquitously active Rosa26‑Cre line, resulted in the deletion of the floxed region in the mouse genome. Mice homozygous for the recombinant allele (HtrA4‑/‑) were viable, fertile and appeared to be normal. The HtrA4 protein was detectable in coronary vessels and in the placenta. However, the loss of HtrA4 affected neither the basic heart nor placental functions. These mice, featuring a conditional null allele of HtrA4, may provide a valuable tool to investigate the role of HtrA4 in development and pathogenesis of coronary heart disease and preeclampsia.