A cryptic species of the big-eared horseshoe bat (Rhinolophus macrotis) was identified in Jiangxi Province, China, based on significant differences in echolocation frequencies and morphology. Consistent with the bimodal distribution of body sizes of R. macrotis specimens obtained from the same cave, we now consider this population to be comprised of two putative species; a large and a small form, with dominant echolocation call frequencies of 49 kHz and 65 kHz, respectively. Cytochrome b sequences of these two phonic forms diverged by 3.16–3.25%, a similar level of divergence to that between the large form and the outgroup, R. rex (3.33–3.77%). These differences strongly suggest that the two phonic forms are distinct species. We also found that the wing loading and aspect ratio of the small form was much lower than that of the large form, suggesting that the small form is capable of foraging in denser forest. Without dietary evidence, the ecological significance of the observed difference in echolocation call frequency between the two forms (16 kHz) remains unknown.