Behavioral isolation between two sympatric and closely related phytophagous ladybird beetles, Henosepilachna vigintioctomaculata and Henosepilachana pustulosa, was studied in laboratory. Mating behavior was investigated using the male-choice test. Males of H. vigintioctomaculata preferred conspecific females to heterospecific females as mates, whereas males of H. pustulosa did not show such preference. Females of H. vigintioctomaculata appeared more fastidious at mating than females of H. pustulosa, irrespective of the species of males. Males of neither species showed tenaciousness following rejection by females. The choice of conspecifics by H. vigintioctomaculata males and a difference in the intensity of rejection between H. vigintioctomaculata females (strong) and H. pustulosa females (weak) result in positive behavioral isolation in both directions of heterospecific matings. Our results thus indicated that positive unilateral mate choice yields bilateral behavioral isolation between these two species.