The purpose of this study was to clarify the relationship among X-box-binding protein 1 unspliced, spliced (XBP1u, s), Forkhead box O1 (FoxO1) and autophagy in the auditory cells under endoplasmic reticulum (ER) stress. In addition, the relationship between ER stress that causes unfolded protein response (UPR) and autophagy was also investigated. The present study reported ER stress induction by tunicamycin treatment that resulted in IRE1α-mediated XBP1 mRNA splicing and autophagy. XBP1 mRNA splicing and FoxO1 were found to be involved in ER stress-induced autophagy. This inference was based on the observation that the expression of LC3-II was suppressed by knockdown of IRE1α, XBP1 or FoxO1. In addition, XBP1u was found to interact with XBP1s in auditory cells under ER stress, functioning as a negative feedback regulator that was based on two important findings. Firstly, there was a significant inverse correlation between XBP1u and XBP1s expressions, and secondly, the expression of XBP1 protein showed different dynamics compared to the XBP1 mRNA level. Furthermore, our results regarding the relationship between XBP1 and FoxO1 by small interfering RNA (siRNA) paradoxically showed negative regulation of FoxO1 expression by XBP1. Our findings revealed that the XBP1-FoxO1 interaction regulated the ER stress-induced autophagy in auditory cells.