The cytochrome b 6 f complex occupies an electrochemically central position in the electron-transport chain bridging the photosynthetic reaction center of PS I and PS II. In plants, the subunits of these thylakoid membrane protein complexes are both chloroplast and nuclear encoded. How the chloroplast-encoded subunits of multi-spanning cytochrome b 6 are targeted and inserted into the thylakoid membrane is not fully understood. Experimental approaches to evaluate the cytochrome b 6 import mechanism in vivo have been limited to bacterial membranes and were not a part of the chloroplast environment. To evaluate the mechanism governing cytochrome b 6 integration in vivo, we performed a comparative analysis of both native and synthetic cytochrome b 6 insertion into purified thylakoids. Using biophysical and biochemical methods, we show that cytochrome b 6 insertion into the thylakoid membrane is a non-spontaneous co-translational process that involves ALB3 insertase. Furthermore, we provided evidence that CSP41 (chloroplast stem-loop-binding protein of 41 kDa) interacts with RNC-cytochrome b 6 complexes, and may be involved in cytochrome b 6 (petB) transcript stabilization or processing.