Doxorubicin (DOX) has dose-dependent toxicity on ovarian follicles (OFs), and the inhibition of different signaling molecules along with the DOX application for enhancing its efficacy can also upsurge this toxicity. Therefore, it is strongly required to explore the mechanism of DOX-induced toxicity in 3D culture systems for protecting the OFs. A microfluidic chip was used to culture a single OF to identify the potential signaling molecules and their combined effects on OFs dynamically. The chip offers better 3D biomimetic microenvironment to the growing OF than 2D culture systems. The OFs cultured on the chip were treated with DOX and the inhibitors of Src, Ca2+, and PIM. Their mutual effects were studied on OFs growth and 17β-estradiol secretion. Besides, the RNA levels of B4GALT2 and UNC5C genes of DOX-exposed OFs were detected by RT-qPCR, and TUNEL staining experiments were conducted to check the OF apoptosis. The results showed that DOX application reduced the OFs growth and hormone secretion and induced apoptosis in the OFs. Moreover, the DOX-induced toxic effects were enriched by Src and PIM inhibition, while reduced by the ER-Ca2+ channel inhibitor. This study specifically demonstrates the synergistic effects of some signaling molecules on DOX-mediated cellular functions of OFs and demands some meditative measures to decipher this toxicity for supporting the female endocrine and reproductive functions. Copyright © 2019. Published by Elsevier Ltd.