An enriched environment can promote adaptability of animals to cope with complex environments. A total of 18-week-old 216 laying hens were randomly divided into 2 groups; of which, one group was housed in conventional battery cages (CC, n = 36), and the others were housed in furnished cages (FC, n = 180). At the end of 64 wk of age, 24 chickens of each group were selected for 4-hour transport treatment. The spleen tissues of laying hens were collected before transportation (
BT
), immediately after transportation, and at 48 h after transportation to detect the expression of the heat shock protective response signaling pathway and inflammatory factors. Serum samples were collected to detect the content of immune cytokines. Transport stress decreased heat shock proteins (
HSP
; including Small HSP, HSP27, HSP40, HSP60, HS70, HSP90, HSP110) in the CC group (
P
< 0.05), whereas there was no significant difference in the expression of HSP (except for Small HSP and HSP40) in the FC group (
P
> 0.05) immediately after transportation. At 48 h after transportation, mRNA levels of HSP (except for Small HSP and HSP40) in the FC group were upregulated, which were higher than those at BT (
P
< 0.05). The changes in HSP60, HSP70, and HSP90 protein levels had similar tendencies. The results showed that housing in furnished cages alleviated the inhibition of expression of HSP in the hens' spleen induced by transport stress. In addition, the hens housed in the FC group had lower expression levels of proinflammatory factors (nuclear transcription factor-kappa B, inducible nitric oxide synthase, cyclooxygenase-2, prostaglandin E synthase, inflammatory cytokines [IL-1β and IL-6], and tumor necrosis factor alpha) (
P
< 0.05). We suggest that the enriched environment can reduce transport stress damage in laying hens and improve resistance to transport stress by regulating expression of heat shock response proteins and inflammatory cytokines.