<jats:title>Abstract</jats:title><jats:sec><jats:title>Purpose</jats:title><jats:p>ML‐2–3 is a novel tetracyclic iridoid derived from <jats:italic>Morinda lucida</jats:italic> Bentham leaves. This compound has anti‐trypanosomal and anti‐leishmanial effects. In this study, the authors investigated effects of ML‐2–3 on in vitro fertilization (IVF) rates, motility, and acrosome reaction of the mouse sperm.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>IVF was performed using sperm from BALB/cByJJcl mice treated with ML‐2–3. Computer‐assisted sperm analysis (CASA) was performed on the sperm of C57BL/6J mice to investigate sperm motility. The effect of ML‐2–3 on the acrosome reaction was examined by observing the fluorescence of sperm labeled with the acrosin‐EGFP transgene.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>ML‐2–3 improved IVF in BALB/cByJJcl mice with low fertilization rates. The optimum concentration of ML‐2–3 in sperm pre‐culture medium was 20 <jats:italic>µ</jats:italic>M, and no significant toxicity of ML‐2–3 was observed in developing embryos at this concentration. ML‐2–3 affected sperm motility but not the acrosome reaction. ML‐2–3 increased the IVF rate of mouse sperm that had been refrigerated for 3 days.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>ML‐2–3 can improve the outcome of IVF and motility without inducing the acrosome reaction in mice. These effects of ML‐2–3 on sperm behaviors are different from those of the similar drugs.</jats:p></jats:sec>