In gene electrotransfer and cardiac ablation with irreversible electroporation, treated muscle cells are typically of elongated shape and their orientation may vary. Orientation of cells in electric field has been reported to affect electroporation, and hence electrodes placement and pulse parameters choice in treatments for achieving homogeneous effect in tissue is important. We investigated how cell orientation influences electroporation with respect to different pulse durations (ns to ms range), both experimentally and numerically. Experimentally detected electroporation (evaluated separately for cells parallel and perpendicular to electric field) via Ca2+ uptake in H9c2 and AC16 cardiomyocytes was numerically modeled using the asymptotic pore equation. Results showed that cell orientation affects electroporation extent: using short, nanosecond pulses, cells perpendicular to electric field are significantly more electroporated than parallel (up to 100-times more pores formed), and with long, millisecond pulses, cells parallel to electric field are more electroporated than perpendicular (up to 1000-times more pores formed). In the range of a few microseconds, cells of both orientations were electroporated to the same extent. Using pulses of a few microseconds lends itself as a new possible strategy in achieving homogeneous electroporation in tissue with elongated cells of different orientation (e.g. electroporation-based cardiac ablation).