Procambarus clarkii is an economically important aquaculture species in China, and pathogens, especially Vibrio parahaemolyticus, have caused significant economic losses in large-scale farming. However, the mechanism of the immune system response to pathogen invasion remains unclear. To delve into the molecular mechanisms behind the immune regulation of the Procambarus clarkii, this study analyzed the transcriptome data of its hemocytes that had been co-cultured with 3-hydroxybutyrate. By analyzing the transcriptome data of hemocytes from P. clarkii co-cultured with 3-hydroxybutyrate, significant differential expression of cytoskeleton genes was observed, suggesting a potential association with immune regulation in P. clarkii. This study annotated and classified cytoskeleton genes and validated their impact on cellular immune regulation. The annotation results showed that out of five cytoskeleton genes, four belonged to the kinesin family (KIF). All contained KISc domains, and they encoded amino acids primarily composed of glutamate and leucine. The secondary protein structure was mainly composed of α-helices. The genes encoding KIFs were generally similar across species, except for variations in the number, type of conserved motifs, and gene structure among kinesin-like-protein genes in different species. Quantitative reverse transcription-polymerase chain reaction results showed that the expression levels of KIFs were higher in the hemopoietic tissues and gills of P. clarkii than in tissues such as eyestalks and muscles (P＜0.05). RNA interference results demonstrated that the optimal knockdown effect was achieved with a dose of 0.10 μg/g body weight of dsKIF11, reducing the expression level of KIF11 to 8.01% of its normal level (P＜0.05). Moreover, compared to the control group, after the knockdown of the KIF11 gene, the phagocytic rates of hemocytes towards green-fluorescence-labeled V. parahaemolyticus and red fluorescent microspheres decreased by 36.26% (P＜0.01) and 41.94% (P＜0.01), respectively. The abovementioned results indicate that KIFs exhibit high conservation and are involved in the phagocytic activity of hemocytes. This finding provides a basis for further investigation of the function and mechanism of KIFs in innate immunity.