This study describes the effects of royal jelly (RJ) on testicular injury induced by nicotine (NIC) in mice. Thirty-six male BALB/c mice were randomly divided into six groups (n = 6). Group 1 received normal saline, group 2 received 100 mg/kgBW/day RJ, groups 3 and 4 received NIC at doses of 0.50 and 1.00 mg/kgBW/day, respectively, and groups 5 and 6 received NIC at doses of 0.50 and 1.00 mg/kg BW/day, respectively, plus RJ. Following 35 days, the serum level of testosterone, histopathological changes, germ cell apoptosis, proliferating cell nuclear antigen (PCNA), malondialdehyde (MDA) content, and antioxidant indexes including total antioxidant capacity (TAC) and catalase (CAT) activity were determined. In addition, the mitochondria-dependent apoptosis was investigated by assessing the Bcl-2, p53, and Caspase-3 mRNA levels expression by reverse transcription-PCR (RT-PCR). Compared to NIC receiving groups, the concomitant administration of RJ could protect the testosterone reduction and histological damages. After RJ treatment, the level of tissue MDA content decreased, while tissue TAC and CAT levels were remarkably increased compared to NIC-exposed groups. Remarkable higher TUNEL-positive germ cells and low PCNA index were observed in NIC receiving groups. Besides, the expression level of Bcl-2 was significantly higher and the p53 and Caspase-3 levels were significantly lower in the RJ co-administration groups than NIC-only receiving groups. Our results confirmed that RJ effectively protects the testis against NIC evoked damages by antioxidant and anti-apoptotic effects involving the up regulation of the antioxidant status, mitochondria-dependent apoptosis pathway prevention, and the proliferating activity improvement.