Speckle-tracking echocardiography can non-invasively estimate myocardial work (MW) to evaluate left ventricular (LV) myocardial systolic function. The present study evaluated whether MW may detect subclinical LV myocardial systolic dysfunction in type 2 diabetes mellitus (T2DM) patients with and without diabetes peripheral neuropathy (DPN). A total of 127 T2DM patients were included in the present study, including 67 T2DM patients with DPN. In addition, 73 sex- and age- matched healthy individuals served as normal controls. The global myocardial work index (GWI), global constructive work (GCW), global waste work (GWW), global positive work (GPW), global negative work (GNW), global work efficiency (GWE) and GCW/GWW were measured and analysed. Furthermore, the differences in MW parameters among normal controls, T2DM patients, and T2DM patients with DPN were analysed. Multiple regression models were built to explore for the independent influencing factors of GWI and GPW values in T2DM patients with DPN. Receiver operating characteristic curve analysis was performed to evaluate the sensitivity and specificity of MW in evaluating subclinical LV myocardial systolic dysfunction in T2DM patients with DPN. The GWI, GCW and GPW of T2DM patients with DPN were significantly decreased compared with those of T2DM patients and normal controls (P < 0.001) and showed a significant decreasing trend overall (P trend < 0.001). GWE and GCW/GWW were significantly decreased in T2DM patients with DPN compared with normal controls (P < 0.05). Although GWW was not significantly different among the three groups, it showed an increasing trend (Ptrend = 0.033). High-density lipoprotein cholesterol (HDL-C) levels were independent influencing factor for decreased GWI (β = 0.21, P = 0.031) and GPW (β = 0.19, P = 0.043) values in T2DM patients with DPN. The combination of the GWI, GCW, GWE, GPW and GCW /GWW had good sensitivity (62.69%) and specificity (89.04%) when evaluating subclinical LV myocardial systolic dysfunction in T2DM patients with DPN. Non-invasive evaluation of LV myocardial work can detect subclinical LV myocardial systolic dysfunction in T2DM patients with and without DPN. DPN has additive deleterious effects on LV myocardial systolic function in T2DM patients. The reduction of HDL-C levels may indicate the occurrence of subclinical LV myocardial systolic dysfunction in T2DM patients with DPN.