HSP110 functions to protect cells, tissues, and organs from noxious conditions. Vasectomy induces apoptosis in the testis; however, little is known about the reason leading to this outcome. The aim of the present study was to evaluate the expression and function of HSP110 in mouse testis after vasectomy. Following bilateral vasectomy, we used fluorescent Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect apoptosis, Western blotting and immunohistochemistry to examine HSP110 expression and localization. Serum antisperm antibody (AsAb) and testosterone were measured by Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Expression of endoplasmic reticulum stress (ERS) sensors and downstream signaling components was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and the phosphorylation of eIF2α and JNK was detected by Western blotting. Vasectomy induced morphologic changes, increased apoptosis in the testis, increased serum AsAb, and decreased testosterone levels. After vasectomy, ORP150 mRNA level was increased first and then decreased, Bcl-2 was decreased, and the expression of HSPA4l, GRP78, GADD153, PERK, ATF6, IRE-1, XBP-1s, Bax, Bak, and caspases and the phosphorylation of eIF2α and JNK were increased. We present that an ER stress-mediated pathway is activated and involved in apoptosis in the testis after vasectomy. HSPA4l and ORP150 may play important roles in maintaining the normal structure and function of testis.