Atrial fibrillation (AF) is the most common cardiac arrhythmia and a major cause of stroke. Abnormal AP propagation is a central cause of AF associated with abnormal gap junction (GJ) conductance [1]. The latter is regulated by alterations to the phosphorylation state of GJ connexin (Cx) proteins, in particular Cx43 and Cx40. We have shown that the serine-threonine protein phosphatases (S-T PPs), calcineurin Aα (CnAα) and PP2A dephosphorylate Cx43-Ser365 and exposes Cx43-Ser368 for PKC phosphorylation, thus reducing GJ conductance. However, the role of S-T PPs and protein kinases (PKs) in AF remains unknown. This study aimed to: 1) investigate changes to Cx43 and Cx40 protein expression and phosphorylation state in tissue from sinus rhythm (SR) and AF patients; 2) measure protein levels of S-T PPs and PKs that may mediate these changes. Human left atrial (LA) appendage biopsies from patients with SR (n=13) or AF (n=13) were snap-frozen in liquid N2 and homogenised. Samples were matched by age, gender and hypertension. Western blots measured the expression of total Cx43 (T-Cx43), Cx43 phosphorylated at Ser368 (Cx43-pSer368), T-Cx40, CnAα, PP2A, PKCε, Inhibitor 1 (I-1), I-1pThr35, PP1 and CaMKII. Values are integrated band-densities normalised to GAPDH (mean±SEM). Differences were tested by ANOVA, with post hoc parametric tests; significance was at p<0.05. The ages of SR and AF groups were (SR 70±2.1 vs AF 72±1.9 yrs, p>0.05). T-Cx43 protein expression significantly decreased in AF (SR 1.73±0.26 vs. AF 1.02±0.07; p<0.001). This was associated with a two-fold increase of Cx43-pSer368 levels. LA T-Cx40 protein expression exhibited two bands (P0 and P1). T-Cx40 was significantly higher in AF (SR 0.90±0.06 vs. AF 1.05±0.05; p<0.05) with a significant increase of the P0 band (SR 0.57±0.07 vs. AF 0.82±0.06; p<0.01). The profile of PPs and PKs were assessed. CnAα and PP2A protein expression were significantly increased in AF, whereas PP1 was unchanged. This was associated with an increase in I-1pThr35, suggesting that the I-1-PP1 pathway is not involved in alteration in Cx phosphorylation associated with AF. PKCε protein expression decreased in AF with a ratio similar to that observed for T-Cx43 (T-Cx43 0.67±0.08 vs PKCε 0.88±0.16). CaMKII protein expression was significantly raised in AF. Thus, in AF there was an increase of T-Cx40 (especially P0) compared with a decrease of T-Cx43 protein levels. The increase of CnAα, PP2A and Cx43-pS368 supports previous data for their role in Cx43-pSer365 dephosphorylation leading to Cx43-Ser368 PKC phosphorylation. Finally, the increased T-Cx40 P0 band may be mediated by enhanced levels of CaMKII. In conclusion, this is the first study to link changes to phosphatase and kinase profiles with alterations to Cx43 and Cx40 phosphorylation state in AF.