Environmental sex determination occurs in many organisms, however the means by which environmental stimuli are translated into endocrine messages remains poorly understood. The N-methyl-ᴅ-aspartate receptor (NMDAR) was evaluated as a candidate neural sensor of environmental signals linking environmental cues to endocrine responses using the crustacean Daphnia pulex. NMDAR agonists, modulators, and antagonists were evaluated for their ability to impact D. pulex male sex determination during early stages of reproductive maturity under conditions that simulated seasonal change. The antagonists MK-801 and desipramine significantly increased male sex determination. Both chemicals are also modulators of serotonergic and noradrenergic systems, thus, we evaluated several modulators of monoamine neurotransmission in an effort to discern which signaling pathways might contribute to male sex determination. Compounds that altered serotonergic signaling also stimulated male sex determination. The involvement of the glutamate and monoamine signaling in male sex determination was supported by the increase in mRNA levels of related receptors and transporters under conditions that stimulate male sex determination. Further, mRNA levels of components of the terminal endocrine pathway responsible for male sex determination were also elevated under stimulatory conditions. Overall, we provide evidence that glutamatergic and serotonergic systems function upstream of the endocrine regulation of male sex determination in early life stage daphnids.