This article aimed to investigate changes in the cytoskeleton of airway smooth muscle cells (ASMCs) in juvenile rats with airway remodeling in asthma. We further investigated the involvement of the RhoA/ROCK signaling pathway mechanism. Rat models of airway remodeling in asthma were established by antigen sensitization with ovalbumin for 2, 4, 6, and 8 weeks. The control group was treated with normal saline instead of ovalbumin. In the intervention group, after 8 weeks of culture, ASMCs were treated with the ROCK-specific inhibitor Y-27632. Immunofluorescence, real-time polymerase chain reaction, and Western blot analyses were used to observe changes in the cytoskeleton (F-actin and α-tubulin) of ASMCs and expressions of RhoA and ROCK. The asthmatic groups had significantly higher average gray values of F-actin in ASMCs compared to the control group (P < 0.01), and these values for the intervention group were significantly lower than those of the 8-week asthmatic group (P < 0.05). Expression levels of the α-tubulin protein in the asthmatic groups were all significantly higher than those of the control group (P < 0.01), and the levels in the intervention group were significantly reduced (P < 0.05). Expressions of RhoA and ROCK mRNA and proteins in all asthmatic groups were significantly higher than those of the control group (P < 0.01). Together, these results demonstrate substantial changes of the ASMC cytoskeleton and abnormal expressions of RhoA and ROCK mRNA and proteins in juvenile rats with airway remodeling in asthma.