We observed a thermotropic phase transition in poly[3,4-dihexyl thiophene-2,2′:5,6′-benzo[1,2-b:4,5-b′]dithiophene] (PDHBDT) thin films accompanied by a transition from a random orientation to an ordered lamellar phase via a nearly hexagonal lattice upon annealing. We demonstrate the effect of temperature-dependent molecular packing on charge carrier mobility (μ) in organic field-effect transistors (OFETs) and photovoltaic characteristics, such as exciton diffusion length (LD) and power conversion efficiency (PCE), in organic solar cells (OSCs) using PDHBDT. The μ was continuously improved with increasing annealing temperature and PDHBDT films annealed at 270 °C resulted in a maximum μ up to 0.46 cm2/(V s) (μavg = 0.22 cm2/(V s)), which is attributed to the well-ordered lamellar structure with a closer π–π stacking distance of 3.5 Å as shown by grazing incidence-angle X-ray diffraction (GIXD). On the other hand, PDHBDT films with a random molecular orientation are more effective in photovoltaic devices than films with an ordered hexagonal or lamellar phase based on current–voltage characteristics of PDHBDT/C60 bilayer solar cells. This observation corresponds to an enhanced dark current density (JD) and a decreased LD upon annealing. This study provides insight into the dependence of charge transport and photovoltaic characteristics on molecular packing in polymer semiconductors, which is crucial for the management of charge and energy transport in a range of organic optoelectronic devices.