Larvae of the flesh fly, Sarcophaga similis exhibit photoperiodic responses to control pupal diapause. Although the external coincidence model is applicable to S. similis photoperiodism, it remains unknown how the circadian clock system integrates day-length information. To explore the mechanisms, we examined the neural circuitry involving circadian clock lateral neurons (LNs) and prothoracicotropic hormone (PTTH) neurons. We also examined the photoperiodic effects on LN-fiber patterns in third-instar S. similis larvae. Immunohistochemistry showed that the clock protein PERIOD and the neuropeptide pigment-dispersing factor (PDF) were co-localized in four cells per brain hemisphere, and we named these PDF-LNs of S. similis. Single-cell polymerase chain reaction of backfilled neurons from the ring gland showed that two pairs of pars lateralis neurons with contralateral axons (PL-c neurons) to the ring gland expressed ptth. Double labeling with immunohistochemistry and backfills revealed that PDF-immunoreactive varicose fibers projected close to fibers from PL-c neurons. short neuropeptide f (snpf) receptor and glutamate-gated chloride channel but not pdf receptor were expressed in PL-c neurons. sNPF and L-glutamate but not PDF acutely inhibited the spontaneous firing activity of PL-c neurons. The number of PDF-immunoreactive varicosities of PDF-LNs in the dorsal protocerebrum was significantly higher under short-day than that under long-day conditions in a time-dependent manner. These results suggest that sNPF and/or glutamate signaling to PTTH neurons and PDF-LNs form a potential neural circuity for the photoperiodic control of pupal diapause and that photoperiod modifies the connectivity strength between PDF-LNs and their post- or pre-neurons in the circuitry.