Aim To study the effect of L-carnitine combined with CT10 regulator of kinase like protein (CrkL) on reducing myocardial cell injury induced by hypoxia/reoxygenation (H/R). Methods H9c2 cells were injured by H/R and treated with L-carnitine. CCK-8, flow cytometry, and Western blot were applied to determine cell proliferation, apoptosis, and nuclear associated antigen Ki67 (Ki-67), proliferating cell nuclear antigen (PCNA),Bü cell lymphoma/lewkmia-2 (Bcl-2), Bcl-2 associated X protein (Bax), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nuclear factor-κB (NF-κB), and CrkL levels, respectively. Cells H9c2 were transfected with pcDNA-CrkL, and treated with H/R or treated with H/R and L-carritine. The above methods were used to detect cell proliferation and apoptosis.Results Compared with the control group, the viability , Ki-67, PCNA, Bcl-2, and CrkL protein expression of H9c2 cells were significantly decreased in the H/R group, and the apoptosis rate, Bax protein level, and the levels of inflammatory factors TNF-α, IL-1β, NF-κB were evidently increased (P＜0.05). Compared with the H/R group, L-carnitine obviously improved the H/R-induced H9c2 cell viability, Ki-67, PCNA, Bcl-2, and CrkL protein expression, and remarkably reduced the apoptosis rate, Bax protein level, and TNF-α, IL-1β, NF-κB levels (P＜0.05). CrkL overexpression dramatically enhanced H/R-induced H9c2 cell viability, Ki-67, PCNA, and Bcl-2 protein expression, while markedly reduced apoptosis rates, Bax protein levels, TNF-α, IL-1β, and NF-κB levels (P＜0.05). Compared with L-carnitine or CrkL overexpression alone, L-carnitine combined with CrkL overexpression clearly increased the viability of H9c2 cells, Ki-67, PCNA, and Bcl-2 protein expression, and distinctly reduced the apoptosis rate and Bax protein level, TNF-α, IL-1β, NF-κB levels (P＜0.05). Conclusion L-carnitine combined with CrkL promotes the proliferation of cardiomyocytes induced by H/R, reduces apoptosis and inflammatory response, and thus protects cardiomyocytes from damage.