<p>In reproduction of Mozambique tilapia (<i>Oreochromis mossambicus</i>), chemical communication mainly relies on urinary steroidal glucuronides (sex pheromones) that dominant males release to prime the final ovulation in pre-ovulatory females and increase reproductive readiness. However, the role of steroids in chemical communication in females has received less attention. Here, we aimed to determine whether 17β-estradiol 3-glucuronate released by pre-ovulatory females functions as a reproductive signal towards males. We investigated the preference of focal males to visual, chemical (female conditioned water), and a combination of both stimuli from pre-ovulatory or post-spawning females, as well as 10^− 9 M 17β-estradiol 3-glucuronate. Compared to the control zone, where no stimuli were present, males were more responsive (time spent near the stimuli, digging behaviour, and urination frequency) in the visual and visual + chemical zones than in the chemical-only zone. Males preferred pre-ovulatory to post-spawning female-conditioned water. Interestingly, the time spent by focal males near the source of 17β-estradiol 3-glucuronate was similar to pre-ovulatory conditioned water. However, there was no significant difference in digging and urine pulses in response to 17β-estradiol 3-glucuronate compared to the control group. We suggest that male tilapia recognise the ovulation status of females using visual and chemical cues, and 17β-estradiol 3-glucuronate is part of the odorant content released by pre-ovulatory females to communicate their reproductive status.</p>