Objective To improve the transformation efficiency of Corynebacterium glutamicum cells with heterogenous plasmid DNA and single-strand DNA (ssDNA) using a methodology based on electro-transformation. Results A semicomplex hypertonic medium was selected with addition of glycine and dl-threonine to weaken cell walls and addition of Tween 80 and isonicotinic acid hydrazide to increase cytoplasmic membrane fluidity. Their contents were optimized by response surface methodology. Cell growth, electro-transformation buffer, and transformation protocol were also optimized. Temporary heating inactivation of the host restriction enzyme showed a significant effect. Finally, a high transformation efficiency of 3.57 +/- 0.13 x 10(7) cfu/mu g DNA of plasmid and 1.05 x 10(6) Str (R) cfu per 10(9) viable cells with a ssDNA was achieved. Conclusion The results shed light on the application in functional genomics and genome editing of C. glutamicum.