Photoperiodism is a biological seasonal timing system utilized to regulate development and reproduction in organisms. The freshwater micro-crustacean Daphnia pulex displays environmental sex determination, the precise physiological mechanisms of which are largely unknown due to the lack of an experimental system to induce female or male offspring production by alterations of the rearing environment. We recently found that D. pulex, WTN6 strain, produces female or male offspring in response to long-day or short-day conditions, respectively. Taking advantage of this system, here we report the photoperiodic response curve for male offspring production, showing 12 hours as natural critical daylength (50% incidence of male-producing mothers), and that male offspring inducibility is highly sensitive to photoperiodic alterations. By using monochromatic light emitting diode (LED) devices, we found that the effective wavelength is red-light (627 nm), which stably induces male offspring production. This suggests that the red-light photoreceptor may be decisive in the primary step of sex determination process in this strain. Our findings provide the first insights into photoperiodism and red-light as key factors in triggering male offspring production in daphnids.